Organic anion-transporting polypeptides (OATPs) are a family of multispecific carriers that mediate the sodium-independent transport of steroid hormone and conjugates, drugs, and numerous anionic endogenous substrates. We investigated whether members of the OATP gene family could mediate fetal-maternal transfer of anionic steroid conjugates in the human placenta. OATP-B (gene symbol SLC21A9) was isolated from a placenta cDNA library. An antiserum to OATP-B detected an 85-kDa protein in basal but not apical syncytiotrophoblast membranes. Immunohistochemistry of first-, second-, and third-trimester placenta showed staining in the cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Trophoblasts that reacted with an antibody to Ki-67, a proliferation-associated antigen, expressed lower levels of OATP-B. OATP-B mRNA levels were measured in isolated trophoblasts under culture conditions that promoted syncytia formation. Real-time quantitative PCR estimated an 8-fold increase in OATP-B expression on differentiation to syncytia. The uptake of [(3)H]estrone-3-sulfate, a substrate for OATP-B, was measured in basal syncytiotrophoblast membrane vesicles. Transport was saturable and partially inhibited by pregnenolone sulfate, a progesterone precursor. Pregnenolone sulfate also partially inhibited OATP-B-mediated transport of estrone-3-sulfate in an oocyte expression system. These findings suggest a physiological role for OATP-B in the placental uptake of fetal-derived sulfated steroids.