The aim of this study was to evaluate the plasma and intracellular pharmacokinetics of raltegravir in HIV-infected patients receiving once-daily raltegravir. Five HIV-infected patients on stable therapy with lopinavir-ritonavir monotherapy whose HIV-1 RNA load was <50 copies/ml were included in this open-label, pilot study. Raltegravir was added to the antiretroviral regimen at a dose of 800 mg once daily from days 0 to 10. On day 10, a full pharmacokinetic profile was obtained for each participant. Raltegravir concentrations in plasma and peripheral blood mononuclear cells (PBMCs) were determined by high-performance liquid chromatography with a fluorescence detector and by liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The values of the raltegravir pharmacokinetic parameters in plasma and PBMCs were calculated by noncompartmental analysis. Raltegravir was well tolerated, and all participants completed the study. No differences in the times to the maximum concentration of raltegravir in plasma or the raltegravir half-lives were observed between plasma and PBMCs. The geometric mean raltegravir maximum concentration, the concentration at the end of the dosing interval, and the area under the concentration-time curve during the dose interval in plasma versus PBMCs were 2,640 ng/ml (range, 887 to 10,605 ng/ml) versus 199 ng/ml (range, 82 to 857 ng/ml) (geometric mean ratio [GMR], 13.30; 95% confidence interval [CI], 3.11 to 56.89; P = 0.003); 89 ng/ml (range, 51 to 200 ng/ml) versus 7 ng/ml (range, 2 to 15 ng/ml) (GMR, 13.21; 95% CI, 3.94 to 44.26; P = 0.001); and 12,200 ng·h/ml (range, 5,152 to 30,130 ng·h/ml) versus 909 ng·h/ml (range, 499 to 2,189 ng·h/ml) (GMR, 13.43; 95% CI, 5.13 to 35.16; P < 0.001), respectively. Raltegravir does not accumulate in PBMCs, with intracellular concentrations being about 1/10 of the concentrations in plasma. Despite once-daily dosing, mean raltegravir concentrations at the end of the dosing interval in plasma and PBMCs exceeded the reported protein-binding-adjusted 95% inhibitory concentration (IC(95)) and IC(50) for wild-type viral strains, respectively.