Saquinavir, a peptidomimetic HIV protease inhibitor, has been shown to be effective in reducing patient viral load and reducing mortality. In this report we investigated whether saquinavir is a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), which may reduce the effective intracellular concentration of the drug. G185 cells, which highly express P-gp, are resistant to saquinavir-mediated cytotoxicity, and co-administration of cyclosporine reversed this resistance. Saquinavir and saquinavir mesylate inhibited basolateral to apical transport of the fluorescent dye rhodamine 123 in a polarized epithelial transport assay, a result that suggests competition of these drugs for the P-gp transporter. Finally, we measured specific, directional transport of saquinavir and saquinavir mesylate in an epithelial monolayer model. Transport in the basolateral to apical direction was 3-fold greater than apical to basolateral flux for both saquinavir and saquinavir mesylate and was blocked by co-incubation with the established P-gp reversal agents cyclosporine and verapamil. These data provide evidence that saquinavir is a substrate for the P-gp transporter and suggest that this protein may affect intracellular accumulation of the drug and contribute to its poor oral bioavailability.