Oatp1, the organic anion transport polypeptide, is an integral membrane protein cloned from rat liver that mediates the uptake of various organic anions such as bromosulfophthalein (BSP) and taurocholate (TCA). Recent studies by others revealed that the thrombin inhibitor, CRC 220, a modified dipeptide, was transported by oatp1. The present study was designed to examine whether another modified peptide, enalapril, an angiotensin-converting enzyme inhibitor, was also a substrate. Transport was studied with enalapril (1 to 800 μmol/L, with [3H]enalapril) in a HeLa cell line stably transfected with oatp1-cDNA under the regulation of a Zn2+-inducible promoter. Noninduced transfected cells (without zinc) that did not express oatp1 failed to take up enalapril. In contrast, cells expressing oatp1 transported enalapril, estrone sulfate (E1S), taurolithocholic acid sulfate (TLCAS), and the glutathione conjugate of BSP (BSPGSH). Uptake of enalapril by oatp1 at 37°C was substantially higher than that at 4°C. The rate at 37°C (uptake rates for induced - noninduced, transfected cells) was linear over 5 minutes and was concentration-dependent, characterized by a Km of 214 ± 67 μmol/L and a Vmax of 0.51 ± 0.15 nmol/min/mg protein. Enalapril uptake was inhibited competitively by BSP (at 1, 5, 10, and 50 μmol/L) and TCA (at 5, 25, and 100 μmol/L) with inhibition constants (Ki) of 2 and 32 μmol/L, respectively. The metabolite enalaprilat was, however, not transported by oatp1. That oatp1 is not a general transporter of anionic compounds was further shown by the lack of transport of harmol sulfate, benzoate, and hippurate. These observations attest to the role of oatp1 as a specific transporter for at least two classes of pharmacologically important peptides.