The objective of this investigation was to differentiate the roles of P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (Mrp2), and CYP3A on saquinavir (SQV) oral absorption. With use of single-pass jejunal perfusion (in situ) and portal vein-cannulated rats (in vivo), SQV absorption was studied under chemical inhibition of Pgp [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2 isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)], Mrp2 [(3-(((3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl) ((3-(dimethylamino-3-oxopropyl)thio)methyl)-thio) propanoic acid (MK571)], and/or CYP3A (midazolam). Plasma concentrations of SQV and related metabolites were analyzed by liquid chromatography-tandem mass spectrometry. When given alone, SQV absorption was extremely low both in situ (F(a) = 0.07%) and in vivo [C(max) = 0.068 microg/ml; area under the curve (AUC) = 6.8 microg x min/ml]. Coadministration of GF120918 boosted SQV absorption by more than 20-fold with decreased variation in AUCs (percent coefficient of variation = 30% versus 100%). In contrast, coadministration of MK571 or midazolam increased SQV absorption only 2- to 3-fold without improving the variation in AUCs. SQV oral absorption was not further improved when it was given with GF120918 and midazolam or with GF120918 and MK571. The current results provide, for the first time, direct and explicit evidence that the low oral absorption of SQV is controlled by a secretory transporter, Pgp, and not by limited passive diffusion owing to its poor physicochemical properties. Pgp-mediated transport is also responsible for the highly variable oral bioavailability of SQV. In contrast, intestinal Mrp2 and intestinal CYP3A appear to play minor roles in SQV oral bioavailability. Given the differential and complex roles of Pgp and CYP3A in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration to avoid therapy-limiting drug-drug transporter and enzyme interactions.