The role of aldehyde dehydrogenase (ALDH) in ex vivo tolerance to transdermal glyceryl trinitrate was explored in rat aorta. ALDH activity, measured by aldehyde-induced NADH formation, was strongly depressed in the tolerant arteries. ALDH inhibitors, chloral hydrate (0.3 mM) and cyanamide (0.1-1 mM) inhibited relaxation to glyceryl trinitrate in non-tolerant and tolerant arteries. The inhibition differed from tolerance in that (a) the glyceryl trinitrate concentration-response curve was sigmoidal cf. biphasic in tolerance, (b) the potentiating effect of nitric oxide synthase (eNOS) inhibition was unchanged cf. increased in tolerance and (c) superoxide inhibited the response cf. no significant effect in tolerant or non-tolerant arteries. Hence, reduced ALDH activity does not account fully for ex vivo tolerance. The discrepancies are consistent with evidence that (a) organic nitrates, unlike chloral and cyanamide, irreversibly inactivate ALDH (hence reduced enzyme saturability can explain the biphasic curve) and (b) eNOS contributes to tolerance by a mechanism independent of glyceryl trinitrate metabolism.