STAT5b is repeatedly activated in rodent liver by the male pattern of intermittent plasma growth hormone (GH) stimulation and is required to maintain the GH pulse-regulated, male-specific pattern of liver gene expression. We presently investigate the interactions between STAT5b and hepatocyte-enriched nuclear factors (HNFs) that contribute to regulation of GH pulse-inducible, male-specific liver cytochrome P-450 (CYP) genes. STAT5 binding sites were identified in the 5'-flank of the adult male-expressed genes CYP2A2 (nucleotides -2255 to -2247), CYP4A2 (nucleotides -1872 to -1864), and CYP2C11 (nucleotides -1150 to -1142). STAT5-DNA complexes were formed by each CYP sequence with nuclear extract from GH pulse-activated male, but not female, rat liver. The CYP2C11 STAT5 site, which is flanked by HNF3 consensus sequences, conferred STAT5b-inducible reporter gene activity in GH-treated HepG2 cells. trans-Activation of the intact CYP2C11 promoter (1.8-kilobase 5'-flank) was strongly induced by the liver nuclear factors HNF1alpha and HNF3beta but, unexpectedly, was inhibited by GH-activated STAT5b. This STAT5b inhibitory effect could be reversed by HNF1alpha and reflects a functional antagonism between STAT5b and HNF3beta, as evidenced by the inhibition of HNF3beta DNA binding and transcriptional activity by STAT5b. HNF3beta, in turn, inhibited STAT5b by a novel mechanism that leads to suppression of GH-inducible STAT5b tyrosine phosphorylation, DNA binding activity, and transcriptional activity. The potential for GH-activated STAT5b to stimulate male-specific liver CYP expression can thus be modulated by HNF3beta, highlighting the complex interrelationship between STAT5b and liver transcription factors controlling expression of GH-regulated CYP genes.