Xanthine oxidoreductase (XOR) is a prominent component of the milk lipid globule, whose concentration is selectively increased in mammary epithelial cells during the transition from pregnancy to lactation. To understand how XOR expression is controlled in the mammary gland, we investigated its properties and regulation by lactogenic hormones in cultured HC11 mammary epithelial cells. XOR was purified as the NAD(+)-dependent dehydrogenase by benzamidine-Sepharose chromatography and was shown to be intact and to have biochemical properties similar to those of enzyme from other sources. Treating confluent HC11 cells with prolactin and cortisol produced a progressive, four- to fivefold, increase in XOR activity, while XOR activity in control cells remained constant. Elevated cellular XOR activity was correlated with increased XOR protein and was due to both increased synthesis and decreased degradation of XOR. Prolactin and cortisol increased XOR protein and mRNA in the presence of epidermal growth factor, which blocked the stimulation of beta-casein synthesis by these hormones. Further, hormonal stimulation of XOR was inhibited by genistein (a protein tyrosine kinase inhibitor) and by PD 98059 (a specific inhibitor of the MAP kinase cascade). These findings indicate that lactogenic hormones stimulate XOR and beta-casein expression via distinct pathways and suggest that a MAP kinase pathway mediates their effects on XOR. Our results provide evidence that lactogenic hormones regulate milk protein synthesis by multiple signaling pathways.