Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.