The ubiquitous transcription factor Oct-1 is involved in the hormonal regulation of the transcription of the major milk protein β-casein through an interaction with the prolactin receptor, the STAT-5, and the glucocorticoid receptor (GR). In this study, this interaction was further demonstrated using Oct-1-deficient cells. In addition, Oct-1 mRNA expression is shown to increase during pregnancy and reach the highest levels during early lactation in mouse mammary gland. In reconstituted COS-7 cells, the endogenous Oct-1 binding activity rapidly increased within 5 min upon the lactogenic hormone treatment, indicating potential post-transcriptional/translational modification of Oct-1 by prolactin and glucocorticoids. Furthermore, STAT-5B was as effective as STAT-5A in the interaction with Oct-1 during hormonal induction, and a GR mutant, which carries mutations at multiple potential phosphorylation sites, functioned similarly to the wild-type GR, indicating that these phosphorylation sites may not be involved in the interaction of GR with Oct-1 on the β-casein gene promoter.