Therapeutic drug monitoring of protease inhibitors (PIs) is usually performed on plasma samples although their antiretroviral effect takes place inside cells. Little is known, however, about the intracellular accumulation and related plasma pharmacokinetics of PIs such as lopinavir/ritonavir (LPV/RTV). Therefore, we studied the plasma and intracellular (cell-associated) steady-state pharmacokinetics of this PI combination in a dosage of 400/100 mg twice daily in a non-randomized cohort of HIV-1-infected individuals. Plasma (0-12 h) and peripheral blood mononuclear cell (PBMC; 0-8 h) samples were drawn during a 12-h dosing interval in 11 subjects. The plasma concentrations versus time curves of LPV and RTV were characterized by an irregular absorption phase showing double-peaks (Cmax) in most subjects and single-peaks in the remaining patients between 1 and 3 h after drug intake. Pre-dose concentrations of both agents in plasma were significantly higher than the concentrations at the end of the dosing interval indicating the presence of a circadian rhythm in their pharmacokinetics. The course of the intracellular concentrations versus time curves appeared to be similar to the plasma concentration curves, with the highest intracellular concentration measured 3 h after drug intake. The intracellular RTV concentrations were higher than reported in vitro EC50 values and might therefore contribute to the antiretroviral effect of LPV/RTV. The median intracellular-to-plasma concentration ratios (interquartile range) were 1.18 (0.74-2.06) and 4.59 (3.20-7.70) for LPV and RTV, respectively. In conclusion, both LPV and RTV accumulate to potential therapeutic concentrations in PBMCs. Irregular absorption and circadian plasma clearance patterns were observed for the PI combination LPV/RTV.