The signal transducer and activator of transcription 5 (Stat5) has been shown to cooperate with some nuclear receptors. However, an interaction has never been demonstrated with the androgen receptor (AR). Given that the PRL-inducible protein/gross cystic disease fluid-15 (PIP/GCDFP-15) is both a PRL-controlled and an androgen-controlled protein, we used its promoter region to investigate the potential interaction between Stat5 and androgen receptor. Dihydrotestosterone or PRL alone slightly modulated or did not modulate the luciferase activity of all reporter gene constructs. In contrast, a maximal increase was observed using the -1477+42 reporter gene construct after exposure to both dihydrotestosterone and PRL. The requirement of half-site androgen-responsive elements and two consensus Stat5-binding elements, Stat5#1 and Stat5#2, was determined by site-directed mutagenesis. Activated Stat5B binds with a higher affinity to Stat5#2 than to Stat5#1. Stat5ADelta749 and Stat5BDelta754 mutants demonstrated that the Stat5 trans-activation domain is involved in the hormonal cooperation. The cooperation depends on the PRL-induced phosphorylation on Tyr(694) in Stat5A and Tyr(699) in Stat5B, as demonstrated using the Stat5AY694F and Stat5BY699F proteins. The use of AR Q798E, C619Y, and C784Y mutants showed that trans-activation, DNA-binding, and ligand-binding domains of AR are essential. Our study thus suggests a functional cooperation between AR and Stat5.