We assessed bovine bone marrow-derived macrophages and monocyte-derived macrophages for expression of inducible nitric oxide synthase (iNOS) activity. Both cell types expressed iNOS activity upon stimulation with Salmonella dublin (S. dublin) or with LPS. A 372-bp fragment of the bovine iNOS mRNA could be amplified by reverse transcription-PCR from mRNA of stimulated macrophages. Cloning and sequencing of the fragment revealed a high degree of homology to human hepatocyte, rat vascular smooth muscle cell, and mouse macrophage iNOS both at the nucleotide (87 to 92%) and amino acid levels (94 to 97%). iNOS mRNA was expressed maximally 6 h after stimulation with S. dublin, whereas maximal nitrite accumulation in supernatants was measured at 24 to 48 h. Significant differences with regard to cytokine regulation of iNOS were observed between murine and bovine macrophages cultured under identical conditions. The most striking difference was the inability of homologous IFN-gamma to induce iNOS both at the level of nitrite production and of mRNA expression in bovine macrophages. TNF-alpha, IL-2, and IL-1 alone or together with IFN-gamma neither induced iNOS nor primed bovine macrophages for enhanced iNOS expression or activity upon stimulation with S. dublin. RhuIL-4, but not rhuTGF-beta, down-regulated S. dublin-induced iNOS activity and mRNA expression in bovine macrophages. Thus, an enzyme with a high degree of homology to rodent iNOS is inducible by stimulation of bovine macrophages with bacteria, but induction and regulation by cytokines occur under more restricted conditions than in rodent macrophages.