Ritonavir-boosted saquinavir (SQV/r) is currently licensed as a twice-daily regimen. Reducing the pill burden with once-daily dosing may improve adherence. Intracellular concentrations of drugs must be related to the clinical efficacy of protease inhibitors. The aims of the study were to determine the cellular and plasma saquinavir and ritonavir concentrations, to determine the half-lives (t(1/2)s) of the drugs in each compartment, and to examine relationships between drug accumulation and lymphocyte subset P glycoprotein (P-gp) expression. Venous blood samples from 12 human immunodeficiency virus-infected patients receiving a hard-gel formulation of SQV/r (1,600/100 mg once daily) were collected at 2, 6, 12, and 24 h after dosing. Peripheral blood mononuclear cells were separated by density gradient centrifugation, and P-gp expression was measured by dual-color flow cytometry. Plasma and intracellular (cell-associated) drug concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. The ratio of the intracellular drug area under the concentration-time curve from 0 to 24 h (AUC(0-24 h)) to plasma drug AUC(0-24 h) was calculated to determine cellular drug accumulation. The median (range) AUC(0-24 h) of saquinavir in plasma was 16.2 (5.7 to 39.3) mg. h. liter(-1), and that in cells was 46.3 (24.7 to 114.6) mg. h. liter(-1). Corresponding ritonavir values were 7.5 (1.5 to 14.6) mg. h. liter(-1) and 10.4 (3.2 to 13.7) mg. h. liter(-1), respectively. The median accumulation ratios of cellular AUC to plasma AUC for saquinavir and ritonavir were 3.31 (range, 1.49 to 6.69) and 1.46 (range, 0.83 to 4.15), respectively. Significant differences between the plasma and intracellular saquinavir t(1/2)s (4.5 h [range, 2.5 to 9.3 h] and 5.9 h [range, 4.0 to 17.7 h]; P = 0.034) and between the plasma and intracellular ritonavir t(1/2)s (4.1 h [range, 2.6 to 8.3 h] and 6.2 h [range, 3.9 to 18.6 h]; P = 0.032) were observed. No relationship was observed between the accumulation of saquinavir or ritonavir and lymphocyte subset P-gp expression. The intracellular t(1/2)s of saquinavir and ritonavir were longer than the plasma t(1/2)s, indicating that intracellular drug may be available at a time when concentrations in plasma are below the minimum effective concentration.