Stat5 is a key intracellular mediator of prolactin signalling and can activate transcription of milk proteins in response to prolactin. Therefore, in animals such as mice where lactation is dependent on prolactin, Stat5 is likely to play an important role in establishing or maintaining lactation in the mammary gland. However, little is known about its role in lactation in the dairy cow. In order to address this, the levels of Stat5a and Stat5b protein, mRNA and Stat5 DNA-binding activity were measured in mammary tissue from mice and cows at different lactational states. In the cow, Stat5a and Stat5b protein and mRNA levels, as well as Stat5 DNA-binding activity were unaltered between pregnancy and established lactation. In contrast, in the mouse Stat5a and Stat5b protein, as well as Stat5 DNA-binding activity were clearly increased during lactation whereas Stat5a and Stat5b mRNA levels were highest during pregnancy as has been previously described. In both species only a minority of the epithelial cell nuclei were Stat5 positive during established lactation. These results suggest that there are significant differences in the biological role of Stat5 in controlling lactation between ruminants and rodents.