This study was designed to determine how pH affects nitric oxide (NO) formation induced by lipopolysaccharide (LPS) in cultured murine macrophages (RAW 264.7). The initial pH of LPS-containing culture media was adjusted to one of eight values (6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.2). After exposure to LPS for eighteen hours, the cultures were harvested for analysis of mRNA, protein, and nitrate/nitrite (stable by-products of NO). Analyses for these substances were performed using semiquantitative RT-PCR, immunoblotting, and colorimetric Griess assays, respectively. We found that acidic culture media favored expression of inducible nitric oxide synthase (iNOS) mRNA. However, alkaline media favored expression of iNOS protein. Our findings suggest that post-transcriptional mechanisms predominate over transcriptional ones in order to regulate pH-mediated effects on NO formation by murine macrophages. The optimal pH for NO formation by iNOS was found in our study to be around 7.2.