Inducible nitric oxide (NO) synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus, we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion, via intra-tracheal instillation of clodronate-liposomes, and subsequent AM-reconstitution, via intra-tracheal instillation of donor iNOS+/+ or iNOS-/- AM. Sepsis was induced by cecal ligation/perforation and ALI assessed at 4hrs: protein leak by the Evans-Blue (EB) dye method, neutrophil infiltration via myeloperoxidase activity (MPO), and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 ± 0.1 vs. 1.4 ± 0.1 µgEB/g lung/min, p<0.05) and MPO activity (37±4 vs. 67±8 U/g lung, p<0.05) compared to non-AM depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9±0.3 µgEB/g lung/min), but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus, septic pulmonary protein leak is absolutely dependent on the presence of functional AM, and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.