While it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), little is known about the effects of diabetes on BK_Ca activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker Diabetic Fatty (ZDF) rat. BK_Ca function in pre-diabetic (5-7 weeks) and diabetic (17-20 weeks) ZDF and Lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly-dissociated myocytes by patch-clamping. Log EC₂₅ values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK_Ca with iberiotoxin (IbTX) in arteries from non- and pre-diabetic but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK_Ca-opener NS1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IbTX-sensitive currents were not enhanced to the extent observed in non-diabetic controls by increasing the Ca²⁺ concentration in the pipette solution or application of NS1619 in myocytes from diabetic animals. Alteration in the expression of BK_Ca beta-1 subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK_Ca of diabetic animals do not significantly oppose vasoconstriction, unlike that of pre-diabetic and control animals. This altered function was related to a reduced Ca²⁺-dependent activation of the channel not involving beta-1 subunits.