We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide (O₂^.-) formation and impairs cAMP-mediated dilation by reducing K_v channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine was observed in RSCAs from DM rats indicating K_v channel impairment. Forskolin increased 4-AP inhibitable K⁺ channel open state probability and whole cell K⁺ current density in coronary myocytes from non DM rats, but had little effect on K⁺ current density in cells from DM rats. Diminished dilation to 8-Bromo-cAMP, forskolin or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic, manganese[III] tetrakis 4-benzoic acid porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of O₂^.- generation by the NADPH oxidase inhibitor, apocynin, in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced O₂^.- formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced O₂^.- formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting K⁺ channel activity.