Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T 4 transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17β-glucuronide (E217βG). Intriguingly, published data suggest that E217βG inhibition of Oatp1c1-mediated T4 transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T4 and E217βG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T4 and E217βG concentrations (100 pM to 300 nM and 27 nM to 200 μM, respectively). Eadie-Hofstee plots derived from these detailed T4 and E217βG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T4 and E217βG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T4 and E 217βG. T4 and E217βG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E217βG transport more strongly than T4 transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.