Abstract: Prolactin (PRL) modulates proliferation in the mammary gland and other tissues, in part through inducing transcription of cyclin D1, a key regulator of G1 phase cell cycle progression. We showed previously that PRL, via Jak2, induces binding of Stat5 to a distal GAS site (GAS1) in the cyclin D1 promoter. However, full promoter activity requires additional regions, and in this paper we explored PRL-induced activity at sites other than GAS1. We defined a second PRL-responsive region spanning −254 to −180 that contains a second GAS site (GAS2) and an Oct-1 binding site. Although mutational analysis indicated independence from GAS2, proximal promoter activity remained Stat5-dependent, suggesting alternative mechanisms. EMSA showed that Oct-1 binds the −254 to −180 region and that PRL decreased Oct-1 binding, leading to increased PRL-responsiveness of the proximal cyclin D1 promoter in multiple cell lines. This suggests a role for Oct-1 in PRL-dependent control of cyclin D1 transcription. [Copyright &y& Elsevier]