Diazoxide and chlorothiazide (0.1–1.5 mM) had a dose-dependent inhibitory effect on the rate of cAMP and cGMP hydrolysis determined in a 500-g supernatant of rat aorta homogenates; both compounds were weaker inhibitors of cAMP and cGMP hydrolysis than theophylline. cAMP and cGMP content of the aorta did not change in the presence of diazoxide or chlorothiazide; diazoxide, however, further increased the isoproterenol-induced rise in cAMP, while chlorothiazide did not. Both benzothiadiazines decreased the maximum tension of the aortic strip induced by serotonin, phenylephrine or potassium. Diazoxide was a stronger and chlorothiazide a weaker inhibitor of the contractile response than theophylline. Comparison of the biochemical and functional effects of diazoxide and chlorothiazide indicates that the inhibitory effect of these compounds on cyclic nucleotide phosphodiesterase does not by itself explain their vasodilating effect.