Objective: As part of the large international, randomized 2NN trial, the pharmacokinetics of nevirapine in once-daily 400 mg and twice-daily 200 mg dosing regimens were investigated. Method: Treatment-naive HIV-1-infected patients were randomized to receive nevirapine 400 mg once daily or 200 mg twice daily, in combination with lamivudine and stavudine. Blood samples were collected at several time-points (day 3, weeks 1, 2, 4, 24, and 48). Differences in pharmacokinetics between once- versus twice-daily dosing were investigated with nonlinear mixed effects modelling (NONMEM). Results: In total, 2,899 nevirapine plasma concentrations were available from 578 patients. Dosage and dosing frequency did not influence clearance or volume of distribution of nevirapine, indicating linear pharmacokinetic behavior of nevirapine whether given as a single daily dose or as divided doses over 24 hours. During steady state, the Cmin was lower (3.26 mg/L vs. 4.44 mg/L; p < .001) and the Cmax was higher (7.88 mg/L vs. 6.55 mg/L; p < .001) in the once-daily arm. However, compared to total variability in nevirapine levels for both treatments, these differences were minor. During steady state, total exposure, measured as AUC24h, was comparable for both regimens (133 mg/L*h vs. 133 mg/L*h; p = .084). Conclusion: The daily exposure to nevirapine (AUC24h) was similar for the 400 mg once-daily and the 200 mg twice-daily dosing regimens. The Cmin of nevirapine is lower and the Cmax of nevirapine is higher for the once-daily regimen as compared to the twice-daily regimen. As a result, 200 mg nevirapine dosed twice daily may be preferred over 400 mg nevirapine dosed once daily.