Smads regulate transcription of defined genes in response to transforming growth factor-beta (TGF-beta) receptor activation. This process involves functional cross-talk of Smads with transcription factors at responsive DNA elements to achieve maximal transcription activation and specificity. TGF-beta has been shown to induce transcription of the germ line (GL) Ig alpha constant region gene and to direct class switching to IgA antibodies. It has been shown that acute myeloid leukemia (AML) transcription factors cooperate with Smad3 to stimulate transcription from the GL Ig alpha constant region gene promoter. We report here that the TGF-beta-induced transcription from this promoter requires DNA binding of cAMP-response element-binding protein (CREB) to the nearby ATF/cAMP-response element site and of Smads to a nearby Smad binding sequence. At these sites, Smad3/4 cooperates with CREB to activate transcription in response to TGF-beta, and disruption of either binding sequence abolished TGF-beta-induced transcription. In addition, AML1 or AML2 also binds to the promoter and cooperates with Smad3/4, and in this way further enhances the TGF-beta-induced transcriptional activation of the GL Ig alpha promoter. Thus, whereas Smad3/4, CREB, and AML family members bind independently to the respective DNA sequences in the GL Ig alpha promoter, functional synergy of Smads with CREB and AML proteins results in maximal TGF-beta-induced transcription.