The lactogenic hormones, i.e., prolactin and glucocorticoids, act in concert to stimulate transcription factors responsible for hormone-dependent milk protein gene expression. In the mammary gland, prolactin activates Stat5a and Stat5b and glucocorticoids activate the glucocorticoid receptor (GR). Immunoprecipitation experiments revealed that in mammary cells, Stat5a, Stat5b, and the GR are physically associated in vivo. The association is not dependent on lactogenic hormone treatment and is evident at all stages of mammary gland development. Immunodepletion experiments indicated that a fraction of GR and Stat5 proteins are not associated, suggesting that there are different intracellular pools of these proteins. Lactogenic hormone treatment of HC11 mammary cells resulted in tyrosine phosphorylation of Stat5a and Stat5b, dimerization, and rapid nuclear translocation of both Stat5 proteins. Following hormone treatment, Stat5a-Stat5b heterodimers were detected by their coimmunoprecipitation. In addition, immunodepletion experiments followed by gel shift analyses revealed the presence of active Stat5a and Stat5b homodimers. In mammary cells, Stat5b homodimers are less abundant than Stat5a homodimers. Although the GR does not bind the Stat5 DNA binding site directly, it could be detected with the Stat5-DNA complex. These results suggest that glucocorticoids affect milk protein gene expression via association of the GR with Stat5. Thus, there is a functional coupling between Stat-dependent and nuclear hormone receptor-dependent gene transcription.