One of the targets of antiretroviral therapy is within cells infected with HIV. In order to improve therapeutic efficacy, it is therefore important that the intracellular pharmacokinetics of drugs, such as nelfinavir mesylate and its active metabolite M8, are studied in addition to plasma pharmacokinetics. Previously, the intracellular accumulation of protease inhibitors has been reported in vivo, displaying the following hierarchy: nelfinavir > saquinavir > ritonavir > indinavir. Multidrug resistance transporters, such as P-glycoprotein (P-gp), may result in a lower intracellular concentration of drug via an efflux mechanism, thus contributing to sanctuary site formation. The objective of this study was to determine concentrations of nelfinavir and M8 in plasma and peripheral blood mononuclear cells from HIV-infected patients, and to ascertain the relationship between intracellular accumulation and lymphocyte P-gp expression. Venous blood samples from 12 HIV-infected patients (viral load <50 copies/ml) receiving nelfinavir (1250 mg twice daily) and dual nucleoside reverse transcriptase inhibitor therapy were collected over a full dosage interval (0, 2, 4, 8 and 12 h). Plasma and intracellular (cell-associated) drug concentrations were measured by HPLC-MS/MS. Drug exposure in plasma and cells was expressed as the area under the concentration-time curve (AUC(0-12h)), derived from non-compartmental modelling. The ratio of intracellular AUC(0-12h)/total plasma AUC(0-12h) was calculated to determine cellular drug accumulation. P-gp expression on lymphocytes was determined by flow cytometry. The median (range) AUC(0-12h) of nelfinavir in plasma and cellular compartments was 21.8 mg x h x l(-1) (5.64-50.8) and 104.6 mg x h x l(-1) (23.1-265.7), respectively. Corresponding values for M8 in plasma and cells were 6.60 mg x h x l(-1) (2.16-17.3) and 19.6 mg x h x l(-1) (5.14-60.8). A ratio of plasma M8/plasma nelfinavir (AUC(0-12h)) and intracellular M8/intracellular nelfinavir (AUC(0-12h)) gave median values of 0.32 and 0.17, respectively. The cellular accumulations [median; (range)] of nelfinavir and M8 were 5.30 (2.28-16.2) and 2.32 (1.01-10.7), respectively. A significant correlation between plasma and intracellular nelfinavir minimum concentration (Cmin) (r2=0.34; P=0.049), but not between plasma and intracellular M8 Cmin was observed. C(0h) concentrations were higher than C(12h) for both nelfinavir and M8. No relationship was observed between nelfinavir or M8 accumulation and lymphocyte cell surface expression of P-gp. This study illustrates that intracellular concentrations were higher than plasma concentrations for both nelfinavir and M8, suggesting lymphocyte accumulation. The mechanism of differential intracellular accumulation of nelfinavir and M8 remains to be elucidated. It may be that affinities for influx transporters or fundamental drug characteristics play a major role in the greater accumulation of nelfinavir than M8.