The aim of the present study was to exclude a potential role of hemoglobin in the formation of nitric oxide (NO) from several nitrovasodilators. NO was measured with a chemiluminescence technique after purging with argon from the aqueous solution. Nitric oxide generation occurred in the absence of hemoglobin or non-heme iron. Sodium nitroprusside and SIN-1 released NO spontaneously. Nitroglycerin produced NO only in the presence of those thiols which are effective co-stimulators of guanylate cyclase. All other thiols degraded nitroglycerin only into nitrite ions without formation of NO. Our results support the role of nitric oxide as terminal activator of guanylate cyclase stimulation by nitrovasodilators.