Inhalational anesthetics inhibit the nitric oxide (NO)-soluble guanylate cyclase signaling pathway in vascular and neuronal tissues and it has been proposed that this inhibition is due to several mechanisms, which include a direct inhibition of NO synthase. To determine the direct interaction of anesthetics with NO synthase, the effects of halothane, isoflurane and enflurane on NO synthase activity of bovine and rat brains and cultured bovine aortic endothelial cells were investigated. Halothane and enflurane at 1% to 3% concentrations produced no significant effect on crude bovine brain NO synthase activity, as measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Similarly, crude rat brain NO synthase activity was not affected by exposure to 1% to 4% halothane or isoflurane. The effects of inhalational anesthetics on the crude bovine brain NO synthase activity were not altered when assayed at two different temperatures (22 degrees C and 37 degrees C). Halothane and isoflurane produced no significant effects on the activity of partially purified rat brain NO synthase at different concentrations of L-[3H]arginine in the reaction mixture. Partially purified endothelial NO synthase, when equilibrated with halothane or isoflurane (0.5-2%), exhibited no significant alteration in enzyme activity. This study suggests that the effects of inhalational anesthetics on NO synthesis in rat and bovine brains and in vascular endothelial cells are not due to their direct interaction with NO synthase.