Protesters shut down roads, causing huge tailbacks into Barcelona in a partial regional strike by pro-independence parties and labour unions.
Protesters blocked roads and train lines across Catalonia on Wednesday, provoking commuter anger in a strike called by a pro-independence union after separatist leaders were detained in Madrid over their divisive secession drive.
Main highways, including Spain's export route to France and the rest of Europe, were cut in about 60 places causing widespread disruption in the region which has been plunged into uncertainty over its now-deposed government's bid to split from Spain.
A few hundred protesters managed to block the tracks at Barcelona's main Sants station.
Authorities said high-speed train links with France were disrupted, with four out of eight daily trains affected.
The independence crisis has further shaken the European Union which is still getting to grips with Britain's decision to leave the bloc, and raised fears of social unrest as well as prolonged disruption to the eurozone's fourth-largest economy.
On Wednesday, Spain's Constitutional Court struck down the declaration of independence made by Catalan lawmakers on October 27 - which led Madrid to dismiss the regional government and assume direct control of Catalonia - declaring the secession bid "unconstitutional and void."
Madrid is organising new regional elections in Catalonia for next month as it tries to stem the fallout from Spain's deepest political crisis in decades.
TRT World's Simon McGregor-Wood has this report.
Main export road cut
Protesters draped huge banners across at least one tunnel in Barcelona, blocking entry, and activists also cut off main roads linking the region of 7.5 million people to France and to the Spanish capital Madrid.
Juan Jose Gil of the National Transport Federation said that the AR-7 highway - "the main transport route for Spanish exports" - was cut in both directions.
The route is used by an average of 9,000 heavy goods vehicles each day, he said.
French authorities said that the border was blocked at the Spanish town of Puigcerda and that other major routes were cut around Figueres, north of Barcelona
Despite the disruptions, the work stoppage was on a smaller scale than a general strike on October 3, which followed Catalonia's banned independence referendum in which 90 percent voted to break from Spain.
Juan Antonio Puigserver, an interior ministry official, said participation in Wednesday's strike had been "minimal" in most sectors.
A judge in Madrid last week ordered eight separatist politicians to be remanded in custody over their secession drive.
Deposed Catalan leader Carles Puigdemont, who fled to Belgium where he is facing extradition back to Spain, on Tuesday criticised the EU for backing Spanish Prime Minister Mariano Rajoy in the crisis.
"Will you accept the result of the Catalan referendum or will you continue to help Mr Rajoy in his coup d'etat?" Puigdemont said in Brussels.
Belgian Prime Minister Charles Michel on Wednesday denied that his government was "in crisis" over Puigdemont's presence, after Flemish separatist members of his coalition government spoke out in support of Catalan independence.
More than 2,000 businesses have moved their legal headquarters out of Catalonia as the turmoil drags on.
Wednesday's walkout was called by the pro-independence CSC union but lacked support from Spain's two largest unions.
Waving pro-independence banners and Catalan flags, demonstrators called for the release of sacked government officials and separatist lobbyists.
In Barcelona, several thousand people gathered outside the Catalan government building, some holding banners reading: "This isn't justice, it's dictatorship".
Fresh elections will be held in Catalonia on December 21 and Rajoy called on Wednesday for "massive participation" in the vote.
Puigdemont has called for a united separatist front to participate, but a former government ally, the leftwing ERC party, on Tuesday ruled out running on the same ticket.
Spain's supreme court in Madrid is due Thursday to resume its hearings of six members of Catalonia's dissolved parliament, including speaker Carme Forcadell, for their role in the secession drive.