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To determine the concentrations of nevirapine (NVP), lamivudine (3TC) and stavudine (D4T) in seminal and blood plasma in HIV-1-infected men.Twelve HIV-1-infected men on NVP-containing regimens including 3TC (n = 8) or D4T (n = 11) provided 23 blood plasma and 22 seminal plasma samples for drug concentration and viral load quantitation. Concentrations of all drugs were assessed by sensitive validated high performance liquid chromatography (HPLC) assays. Blood plasma and seminal plasma viral loads were measured using nucleic acid sequence-based amplification (NASBA). Samples were grouped according to time after drug ingestion, 0–2, 2–4, 4–8 and 8–12 h. For matched seminal and blood plasma samples, obtained within 1 h of each other, a seminal : blood plasma ratio was calculated.The concentration of NVP in seminal plasma appeared to mirror the concentrations in blood plasma. Absolute median seminal plasma NVP concentrations at 0–2, 2–4, 4–8 and 8–12 h were 3.1 μg/ml (range 1.7–4.89), 2.68 μg/ml (2.5–3.9), 2.5 μg/ml (2.3–2.7) and 3.09 μg/ml (1.3–9.1). The median seminal : blood plasma ratios for the four time periods were 0.54 (range 0.34–0.85), 0.83 (range 0.43–1.08), 0.53 (0.48–0.59), and 0.61 (0.59–0.78). 3TC and D4T appeared to reach concentrations in seminal plasma of a similar magnitude or higher than concentrations in blood plasma. The median seminal plasma viral load for all patients was less than 800 copies/ml (range < 800–11 000). The median blood plasma viral load was less than 400 copies/ml (< 400–1100).NVP reaches concentrations in the semen approximately 60% of those in the blood plasma throughout the 12 h dosing period. In a smaller dataset, 3TC and D4T concentrations in blood plasma and seminal plasma were similar. These data may well have implications for the evolution of drug-resistant virus within the genital tract.