CYP3A activity is induced by ~2-fold during the 3(rd) trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from pre-menopausal female donors (n=2) with the physiologic (unbound, 1X-total) and the 10X-total 3(rd) trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5 and the respective hormone receptors (growth hormone receptor, GHR; glucocorticoid receptor, GR; estrogen receptor alpha, ERα). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/GH and cortisol induced CYP3A activity and expression significantly more than cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the 3(rd) trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the 1(st) and 2(nd) trimester, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.