Levels of the type IIbeta regulatory subunit (RIIbeta) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RIIbeta in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RIIbeta can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RIIbeta-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RIIbeta resulted in nuclear localization of a portion of the ectopically expressed RIIbeta. In vitro and in vivo analyses revealed a novel, specific interaction between RIIbeta and CREB that mapped to the N-terminal 135 aa of RIIbeta. In functional studies, RIIbeta inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RIIbeta completely abrogated inhibition. Additionally, RIIbeta suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RIIbeta can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RIIbeta in systemic lupus erythematosus T cells.