Several recent reports have investigated the possibility that nitric oxide (-NO) can regulate prostaglandin H synthase (PHS) activity. The potential significance of -NO regulation of PHS is considerable, when one considers the numerous important biological processes that are influenced by PHS. In this study, we used microsomal and purified PHS to investigate the direct effect of -NO and -NO-generating compounds on PHS catalytic activity. We found that -NO neither significantly inhibited nor enhanced prostaglandin (PG) formation, despite the fact that -NO stimulated PHS peroxidase activity. We also investigated the effect of -NO and -NO generators on PHS product, protein, and mRNA levels in the RAW264.7 murine macrophage cell line. We found that -NO or -NO generators had little or no effect on PG formation, PHS expression, or PHS mRNA expression in unstimulated RAW264.7 cells. The same results were obtained with macrophages that were stimulated by 18 h pretreatment with lipopolysaccharide, a known inducer of PHS-2 in macrophages. These data clearly indicate that -NO acts as a cosubstrate for PHS peroxidase. However, -NO does not enhance or inhibit either cyclooxygenase activity or expression of PHS in the model systems used in this study.