Signal transducer and activator of transcription (Stat)5 has been implicated in the signal transduction pathways of several factors that are lactogenic or galactopoeitic in mammary cells, including prolactin, GH, and IGF-I. Data from cell or explant culture support the concept that Stat5 may represent part of a common route by which different extracellular signals converge and are transduced into the cell. There are few data on Stat5 activity and level in vivo, and we set out to determine whether physiological stimuli of milk synthesis, including GH, GH-releasing factor, and milking frequency, would be associated with alterations in Stat5 activity or protein. We measured Stat5 DNA binding activity using electrophoretic mobility shift assay and Stat5 protein by Western blot in bovine mammary tissue obtained by biopsy or slaughter. Stat5 activity was absent in nonlactating, nonpregnant cows and was present in late pregnancy and throughout lactation. Stat5 activity varied considerably among cows at similar stages of lactation. Mammary Stat5 activity and protein were determined in hormone-treated lactating cows and mammary quarters of cows milked at different frequencies. Infusion of GH and GH-releasing factor for 2 mo significantly raised levels of milk production and depressed mammary Stat5 activity without influencing Stat5 protein abundance. Mammary Stat5 was also influenced by milking frequency; once-daily milking reduced milk production, Stat5 activity, and protein abundance compared with twice-daily milking. Analysis of mammary Stat5 in relation to milk protein concentration in pooled data from lactating cows indicated that Stat5 activity was correlated (r = 0.505, P < 0.05) with average milk protein concentration and not related to milk protein yield (P > 0.05). These results show that both Stat5 protein and Stat5 activity are modulated by different physiological signals in vivo and suggest that Stat5 lies within in the zone where signal transduction cascades from a variety of factors are convergent. Further work is required to clarify the role of Stat5 in relation to other factors in regulation of milk protein gene expression.