The interaction of GH with its receptor has been shown to lead to the phosphorylation of the signal transducer and activator of transcription (STAT) family of transcription factors. We demonstrate here that GH activates the tyrosine phosphorylation of STAT5 in the human IM-9 lymphocyte cell line. Western blotting indicates that GH also activates STAT5 in human embryonic kidney cells (293), which stably express the rabbit GH receptor. Although it has been shown previously that GH activates both STATs 1 and 3 in the 3T3-F442A mouse preadipocyte cell line, we demonstrate that GH also activates STAT5 in these cells. Using electrophoretic mobility shift assay, we examined the interaction of proteins with DNA elements containing consensus STAT-binding sequences. Proteins prepared from GH-treated 3T3-F442A cells bound to the c-sis inducible element of the human c-fos gene (m67 SIE), whereas proteins from GH-treated IM-9 cells did not. However, proteins from GH-treated IM-9 cells did interact with oligonucleotides containing either an interferon response element or the lactogenic hormone-responsive region. Treatment of IM-9 cells with interferon-gamma also induced protein interactions with these elements although the complexes were distinctly different than those seen with GH treatment. Using STAT-specific antibodies, we demonstrate that the GH-induced DNA-protein complex formed with the lactogenic hormone-responsive region contained STAT5, while the interferon-gamma-induced complex contained STAT1. These results implicate STAT5 as a downstream mediator of GH action in IM-9 cells. We report here the cloning of two forms of STAT5, STAT5A and STAT5B, from an IM-9 cDNA library. Northern blot analysis demonstrated multiple-forms of STAT5 mRNA in IM-9 cells.