We report that the functional interaction between cyclin D1 and the estrogen receptor (ER) is regulated by a signal transduction pathway involving the second messenger, cyclic AMP (cAMP). The cell-permeable cAMP analogue 8-bromo-cAMP caused a concentration-dependent enhancement of cyclin D1-ER complex formation, as judged both by coimmunoprecipitation and mammalian two-hybrid analysis. This effect was paralleled by increases in ligand-independent ER-mediated transcription from an estrogen response element containing reporter construct. These effects of 8-bromo-cAMP were antagonized by a specific protein kinase A (PKA) inhibitor, indicating that the signaling pathway involved was PKA dependent. Further, we show that culture of MCF-7 cells on a cellular substratum of murine preadipocytes also enhanced the functional interaction between cyclin D1 and ER in a PKA-dependent manner. These findings demonstrate a collaboration between cAMP signaling and cyclin D1 in the ligand-independent activation of ER-mediated transcription in mammary epithelial cells and show that the functional associations of cyclin D1 are regulated as a function of cellular context.