Pregnancy alters hepatic drug metabolism, but the responsible factors and underlying mechanism remain to be identified. Physiological changes accompanying pregnancy are potentially responsible for the changes, such as rising concentrations of pregnancy-specific somatotropins: prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-V). Whether these hormones influence hepatic drug metabolism in humans is unknown. In this study, we evaluated the effects of PRL, PL, and GH-V on expression of major hepatic CYP enzymes. To this end, freshly isolated human hepatocytes (from 3 different donors) were treated with vehicle (phosphate buffered saline), PRL (150 ng/mL), PL (6 μg/mL), or GH-V (20 ng/mL) for 72 hr, and mRNA expression levels of CYP 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and Na+-taurocholate cotransporting polypeptide (NTCP; used as positive control based on a previous report in rat hepatocytes) were determined by quantitative real-time PCR. PL increased mRNA expression level of CYP2E1 (2-fold) in the hepatocytes while PL did not affect expression of other CYP isoforms. PRL and GH-V had negligible effects on expression of all CYP isoforms tested. Interestingly, NTCP expression was not influenced by any of the hormones, suggesting potential interspecies differences in somatotropin pharmacology between rats and humans. Taken together, these results indicate that except PL, pregnancy-specific somatotropins are unlikely responsible for altered drug metabolism during pregnancy. Further study is needed to elucidate regulatory mechanism underlying upregulation of CYP2E1 expression by PL.