Although it is the best characterized in vitro model of GH (GH) action, the mechanisms used by GH to induce differentiation of murine 3T3-F442A preadipocytes remain unclear. Here we have examined the role of three transcriptional regulators in adipogenesis. These regulators are either rapidly induced in response to GH (Stra13, Stat3) or of central importance to GH signaling (Stat5). Retroviral transfection of 3T3-F442A preadipocytes was used to increase expression of Stra13, Stat3 and Stat5a. Only Stat5a transfection increased the expression of adipogenic markers PPAR, C/EBP and aP2 in response to GH, as determined by quantitative RT-PCR. Transfection with constitutively active Stat3 and Stat5a revealed that constitutively active Stat5a but not Stat3 was able to replace the GH requirement for adipogenesis. Constitutively active Stat5a but not Stat3 was able to increase the formation of lipid droplets and expression of -glycerol phosphate dehydrogenase toward levels seen in mature adipocytes. Constitutively active Stat5a was also able to increase the expression of transcripts for C/EBP to similar levels as GH, and of C/EBP, PPAR and aP2 transcripts to a lesser extent. An in vivo role for GH in murine adipogenesis is supported by significantly decreased epididymal fat depot size in young GH receptor-deleted mice, before manifestation of the lipolytic actions of GH. We conclude that Stat5 is a critical factor in GH-induced, and potentially PRL-induced, murine adipogenesis.