Biosynthesis of nitric oxide (NO) requires L-arginine and molecular oxygen. Although the apparent Km values for L-arginine of NO synthase isoforms have been reported, the apparent Km values for oxygen are unknown. Low oxygen tension has been shown to attenuate NO synthase activity and NO-dependent vascular relaxation. We investigated the effect of different concentrations of oxygen on NO synthase activity of bovine brain, cultured bovine aortic endothelial cells and RAW 264.7 macrophages. The apparent Km values for oxygen were 23.2 +/- 2.8, 7.7 +/- 1.6 and 6.3 +/- 0.9 microM for the brain, endothelial and macrophage NO synthases, respectively. This suggests that pathophysiological conditions involving a decrease in tissue oxygen concentration may attenuate NO production.