In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.