To compare intracellular and plasma etravirine concentrations when etravirine was given at 200 mg/12 h versus 400 mg/24 h and to evaluate whether the results would support once-daily dosing.This was an open-label sequential study in which eight patients on protease inhibitor (PI)-sparing regimens containing etravirine were included. Full pharmacokinetic profiles were performed while on 200 mg of etravirine/12 h and after switching to 400 mg of etravirine/24 h. Intracellular and plasma levels were determined by liquid chromatography coupled with mass spectrometry. Pharmacokinetic parameters were calculated by non-compartmental analysis and compared by geometric mean ratios (GMRs) using 200 mg of etravirine/12 h as the reference group.ClinicalTrials.gov NCT01121809.The geometric mean (GM) for etravirine AUC(0-τ) (5602 versus 5076 ng · h/mL, GMR 0.91), C(max) (403 versus 495 ng/mL, GMR 1.23) and C(min) (139 versus 102 ng/mL, GMR 0.74) were similar with both dosing schedules at the intracellular level. In plasma, the GMRs for AUC(0-τ), C(max) and C(min) were 1.31, 1.76 and 0.99, respectively. The mean intracellular penetration, evaluated as intracellular and plasma AUC(0-τ) ratios, was 81% when etravirine was dosed twice daily and 56% with once-daily dosing.Our results show that intracellular etravirine levels were similar with both dosing regimens in patients with PI-sparing regimens, while etravirine plasma AUC(0-τ) and C(max) were 30% and 76% higher with the once-daily regimen, respectively. Thus, a once-daily dosing regimen is supported not only by plasma etravirine pharmacokinetic profiles but also by intracellular levels.