Growth hormone (GH) and prolactin (PRL) exert long-term effects on cellular metabolism, growth, and development through changes in gene expression and protein biosynthesis that are initiated by hormone binding to specific cell-surface receptors. Recent studies have demonstrated that ligand-induced activation of both GH and PRL receptors leads to the tyrosine phosphorylation of multiple intracellular proteins by the identical non-receptor tyrosine kinase, JAK2. We have shown previously that in vivo administration of human recombinant GH rapidly stimulated the inducible transcription factors, Stats1, 3, and 5, and acutely altered gene transcription in the liver. Because human GH can bind to both lactogenic and somatogenic receptors with high affinity, in this study we have addressed the question of specificity of the hormonal response by examining the early nuclear events following a single injection of rat GH or rat PRL to hormone-deficient hypophysectomized female rats. We find that PRL stimulated tyrosine phosphorylation of Stat5, induced nuclear protein binding to the GH-responsive element of the serine protease inhibitor (Spi) 2.1 promoter, and activated Spi 2.1 gene expression. These acute actions of rat PRL were modest compared to the effects of rat GH. GH treatment induced tyrosine phosphorylation of several hepatic nuclear proteins, activated Stats1, 3, and 5, stimulated Spi 2.1 gene expression, and inhibited albumin gene transcription. All of the effects of rat GH paralleled responses to human GH that we have measured previously. Based on these results, it is likely that most of the actions of human GH in the liver are mediated by the GH receptor rather than by the PRL receptor. The diminished response to PRL may be secondary to the high density of short PRL receptor isoforms in the liver, which do not participate effectively in ligand-induced signal transmission.