Transforming growth factor (TGF)-beta1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-beta1 down-regulates cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A promoter into mink lung epithelial cells in the absence and presence of TGF-beta1. The TGF-beta1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolished down-regulation. Surprisingly, although TGF-beta1 treatment for 24 h markedly decreased cyclin A promoter activity, it did not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic AMP-responsive binding protein (CREB). However, we detected 90 and 78% reductions (by Western analysis) in phosphorylated CREB and ATF-1, respectively, in mink lung epithelial cells treated with TGF-beta1. TGF-beta1-induced down-regulation of cyclin A promoter activity was reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A). These data indicate that TGF-beta1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.