Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12 as well as 10 transmembrane domain structures. Oatp1a1 has 4 potential N-linked glycosylation sites. Which of these sites are actually glycosylated, and their influence on transport function has not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10 from a 12 transmembrane domain model. To map glycosylation sites, the asparagines at the each of the 4 N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12 transmembrane domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, while the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity, but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well-expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.