Rat Oatp1 (Slc21a1) is an organic anion transporting polypeptide that is believed to function as an anion exchanger. In an attempt to characterize its mechanism of transport, this protein was expressed in the yeast S. cerevisiae under the control of the GAL1 promoter. Although Oatp1 protein was present at high levels in isolated S. cerevisiae secretory vesicles, the polypeptide appeared to have minimal post-translational modifications and failed to exhibit taurocholate transport activity. The apparent molecular mass of Oatp1 in yeast was similar to that of the unmodified protein, ~62 kDa, whereas in liver plasma membranes Oatp1 has an apparent molecular mass of ~85 kDa. To assess whether the underglycosylation of Oatp1 in yeast contributed to the lack of functional activity, Oatp1 was expressed in Xenopus leavies oocytes in the presence and absence of tunicamycin, an inhibitor of glycosylation. In oocytes treated with tunicamycin the apparent molecular mass of Oatp1 was decreased from ~72 kDa to ~62 kDa, and transport activity was nearly abolished. Mutations to the four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed that there was a cumulative effect on the function of Oatp1, leading to the total loss of taurocholate transport activity when all glycosylation sites were removed. The apparent molecular mass of the quadruple mutant was ~ 62 kDa, confirming that these four asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction, yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.