We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).