17alpha-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K(m) (Michaelis constant) model (K(m1) = 220 nM; K(m2) = 15.5 microM). Uptake was inhibited (approximately 90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na(+)/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K(m) values (87 nM and 141 microM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single K(m) kinetics (10.7 and 2.6 microM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (K(m1) = 2.9 and K(m2) = 307 microM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.