Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when NO-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K⁺), and hydrogen peroxide (H₂O₂) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild type (WT, normal), db/db (advanced type 2 diabetic) mice and db/db mice null for TNF (db^TNF-/db^TNF-). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N^G-nitro-L-arginine-methyl ester (L-NAME, 10 µmol/L) and prostaglandin synthase inhibitor indomethacin (Indo, 10 µmol/L)] was diminished after administration of catalase [an enzyme that selectively dismutates hydrogen peroxide (H₂O₂) to water and oxygen, 1,000 U/ml]; administration of the combination of charybdotoxin (CTX, nonselective blocker of IK_Ca, 10 µmol/L) and apamin (APA, selective blocker of SK_Ca, 50 µmol/L) also attenuated EDHF-induced vasodilation, but the inhibition of epoxyeicosatrienoic acids synthesis [EETs; 14, 15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE); 10 µmol/L] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after inhibition of potassium (K⁺) channel, EETs synthesis, or H₂O₂ production. Our molecular results indicate that mRNA and protein expression of interlukin-6 (IL-6) were greater in db/db vs WT and db^TNF-/db ^TNF- mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg/ml/kg, i.p. for 3 days) attenuated IL-6 expression in db/db mice. Incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of L-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of L-NAME and Indo in db/db mice. In db^TNF-/db^TNF- mice, EDHF-induced vasodilation was greater and comparable to controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6 induced endothelial dysfunction by activation of H₂O₂ or a K⁺ channel in type 2 diabetes.