Our objective was to study the steady-state plasma and intrapulmonary orally administered ethambutol concentrations in healthy volunteers and subjects with AIDS. Ethambutol (15 mg/kg of body weight) was administered orally once daily to 10 men with AIDS, 10 healthy men, 10 women with AIDS, and 10 healthy women. The mean (±standard deviation [SD]) CD4 cell count for the 20 subjects with AIDS was (350 ± 169) × 10⁶ cells per liter. Blood was obtained for drug assay 2 h after the last dose and at the completion of bronchoalveolar lavage, performed 4 h after the last dose. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) was calculated by the urea dilution method. The total number of alveolar cells (AC) was counted in a hemocytometer, and differential cell counting was performed after cytocentrifugation. Ethambutol was measured by a new, sensitive and specific liquid chromotography-mass spectrometry method. The presence of AIDS, as defined in this study, or gender was without significant effect on the concentrations of ethambutol in plasma at 2 or 4 h or in ELF at 4 h following the last dose. Plasma drug concentrations (mean ± SD) at 2 and 4 h were 2.1 ± 1.2 and 2.1 ± 0.8 µg/ml, respectively, and both values were not significantly different from the concentration of ethambutol in ELF at 4 h (2.2 ± 1.1 µg/ml). The concentration of ethambutol was significantly greater in AC in all four groups (range, 44.5 ± 15.6 to 82.0 ± 39.4 µg/ml) than in ELF or plasma and was approximately 30 to 240 times the reported MIC for ethambutol-susceptible strains of Mycobacterium tuberculosis. The AC ethambutol concentration (mean ± SD) in the smoking women (97.2 ± 32.1 µg/ml) was more than twice the concentration in all other nonsmoking subjects (45.2 ± 16.8 µg/ml) combined (P < 0.05). Two- and 4-h concentrations of ethambutol in plasma were not affected by AIDS status or gender. The high AC/plasma and AC/ELF concentration ratios suggest that substantial antimycobacterial activity resides in these cells. The data confirm earlier observations of active transport ex vivo of ethambutol into pulmonary macrophages.