Glycogen synthase kinase 3 beta (GSK3b) is a serine/threonine protein kinase. Transgenic mice expressing a constitutive active GSK3b in the heart were protected against isoproterenol-induced hypertrophy, suggesting antagonism of GSK3b and PKA-mediated signal transduction. To investigate GSK3b dependent signalling we identified GSK3b-associated protein complexes via Tandem Affinity Purification (TAP). Therefore, we fused a TAP-Tag to the C-terminus of GSK3b and performed a two-step purification of the recombinant GSK3b and its interacting partners under native conditions. To analyse whether the protein interactions are depended on the catalytic activity of GSK3b we constructed a constitutively active and a kinase dead (KD) mutant of GSK3b-Tag. TAP of GSK3b-Tag and identification of copurified proteins by MS-analysis resulted in the identification of GSK3b associated proteins which in part are depended on GSK3b catalytic activity. In HL1-cardiomyocytes we found complexes of the active GSK3b e.g. with Acetyl-CoA-carboxylase and Axin, whereas the inactive protein interacted with Calnexin and HSP90co-chaperone Cdc37. In HEK293-cells the inactive form was linked to CAD protein and to Tubulin b whereas the active GSK3b e.g. interacted with AKAP220 and formed a prominent complex with Proteinkinase A I alpha. Treatment of cells or the isolated complexes with the PKA stimulator 8-Br-cAMP induced dissociation of the PKA catalytic but not the regulatory subunit and increased P-GSK3b levels demonstrating that GSK3b is a substrate of PKA. In contrast, the KD mutant did not interact with PKA.GSK3b forms multiple protein complexes depending on its catalytic activity. A regulated interaction with PKA may form the basis for the observed antagonism of PKA and GSK3b.