To understand the role of nitric oxide (NO) in the regulation of cellular metabolism in peritoneal macrophages under physiological low oxygen tension, its effect on the respiration and energy metabolism was examined with casein-induced peritoneal macrophages from the rat. Intraperitoneal injection of casein transiently induced peritoneal infiltration of neutrophils (peaked on day 1) followed by the migration of macrophages that peaked on day 2. Western blotting analysis using antibodies against inducible type of NO synthase (iNOS) revealed that macrophages appeared in the peritoneal cavity during an early stage (approximately day 2) but not during the late stage (day 3 approximately) of inflammation expressed iNOS and generated substantial amounts of NO by a mechanism that was inhibited by N-iminoethyl-L-ornithine (NIO), a specific inhibitor of iNOS. Although NO reversibly but strongly inhibited the respiration of macrophages from both stages particularly under physiologically low oxygen tension, NIO markedly enhanced the respiration of macrophages obtained from the early period but not from the late period of inflammation. The ATP level in the macrophages from the late period but not from the early period was markedly decreased by NO. Biochemical analysis revealed that the glycolytic activity in the macrophages obtained from the early period was significantly higher than that from the late period of inflammation. These results indicate that significant fractions of cellular ATP in iNOS-positive peritoneal macrophages are synthesized by the increased activity of glycolysis particularly under physiological low oxygen tensions where the mitochondrial respiration is strongly inhibited by endogenously generated NO by macrophages and neutrophils.