Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-kappaB (NF-kappaB) transcription factor since exposure of macrophages to low environmental pH both increased NF-kappaB binding activity in the nucleus and enhanced NF-kappaB-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-kappaB translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor alpha (TNFalpha). Indeed, 1) elevated TNFalpha bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNFalpha antibody impaired both NF-kappaB activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-kappaB.