In human primary T cells, the type II isozyme of protein kinase A (PKA-II) is localized to cytoskeletal elements or organelle membranes. Stimulation of T cells via the T cell receptor/CD3 complex or by addition of the cAMP analog, 8-Cl-cAMP, activates PKA-II, resulting in nuclear translocation of the RIIbeta-subunit from the cytosol and apparent RIIbeta DNA-binding. In current experiments, we demonstrated that recombinant RIIbeta forms a heterodimer with recombinant CREB, a nuclear transcription factor, as shown both by EMSA and immunoprecipitation/ immunoblotting. We found no evidence of direct binding of RIIb to c-fos-defined CRE oligonucleotides by EMSA. Although the RIIbeta-CREB heterodimer binds to the c-fos cAMP response element (CRE), phosphorylation of both RIIbeta and CREB by PKA enhances binding to c-fos CRE oligos. In vivo, phorbol myristate acetate (PMA)-induced synthesis of c-Fos protein is inhibited by DNA-binding of RIIbeta-CREB complexes. Taken together, these data suggest that, in addition to its primary function as an inhibitor of catalytic-subunit activity, the RIIbeta-subunit also acts as a transcription factor that can modulate the activity of the c-fos promoter. Therefore, we propose that RIIbeta may be a transcriptional repressor of c-fos.