STATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like progesterone receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a beta-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21(WAF1) and c-fos promoters. These data demonstrate the convergence of progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland.