Abstract Background The majority of the genes involved in the inflammatory response are highly conserved in mammals. These genes are not significantly expressed under normal conditions and are mainly regulated at the transcription and prost-transcriptional level. Transcription from the promoters of these genes is very dependent on NF-κB activation, which integrates the response to diverse extracellular stresses. However, in spite of the high conservation of the pattern of promoter regulation in κB-regulated genes, there is inter-species diversity in some genes. One example is nitric oxide synthase 2 (NOS-2), which exhibits a species-specific pattern of expression in response to infection or pro-inflammatory challenge. Results We have conducted a comparative genomic analysis of NOS-2 with different bioinformatic approaches. This analysis shows that in the NOS-2 gene promoter the position and the evolutionary divergence of some conserved regions are different in rodents and non-rodent mammals, and in particular in primates. Two not previously described distal regions in rodents that are similar to the unique upstream region responsible of the NF-κB activation of NOS-2 in humans are fragmented and translocated to different locations in the rodent promoters. The rodent sequences moreover lack the functional κB sites and IFN-γ response sites present in the homologous human, rhesus monkey and chimpanzee regions. The absence of κB binding in these regions was confirmed by electrophoretic mobility shift assays. Conclusion The data presented reveal divergence between rodents and other mammals in the location and functionality of conserved regions of the NOS-2 promoter containing NF-κB and IFN-γ response elements.