We have investigated the function of endothelial Ca²⁺-activated K⁺ channels in arteries of the Zucker Diabetic Fatty (ZDF) rat, a model of Type II diabetes. Responses were examined in 17-20 week old male ZDF (fa/fa) animals with approximately 7-10 weeks of established diabetes (glucosuria) together with age-matched lean (fa/+ and +/+) control animals. Animals were killed by a rising CO₂ concentration followed by cervical dislocation. Sharp microelectrode recordings of smooth muscle membrane potential were obtained from small (< 300μm outer diameter) mesenteric arteries perfused with Krebs solution containing 10μM indomethacin plus 100μM L-nitroarginine. Resting membrane potentials were similar in control and diabetic arteries (-54.2±0.5mV and -54.7±0.5mV, respectively; mean±sem, n=12). Smooth muscle hyperpolarizations elicited by 1 and 10μM ACh were significantly smaller in arteries from diabetic compared to control animals (15.2±1.1mV compared to 19.6±0.6mV response to 10μM ACh, p=0.003 by t-test, n=12). Responses in both groups were completely inhibited by a combination of 100nM apamin plus 10μM TRAM-39. Application of apamin alone revealed an IK_Ca-mediated component of the hyperpolarization that was comparable between groups (~13mV on exposure to 10μM ACh). However, in the presence of TRAM-39 alone, the SK_Ca-mediated hyperpolarization was reduced in diabetic compared to control animals (response to 10μM ACh: 9.6±1.2mV versus 17.9±1.2mV, respectively, p<0.001, n=6). The similarity between the IK_Ca-mediated responses in control and diabetic groups suggests that the impairment mechanism does not involve receptor signalling and global Ca²⁺ mobilisation. The deficits in hyperpolarizing responses to ACh, i.e. overall and apamin-sensitive components, were not observed in pre-diabetic 5-6 week old animals. These results demonstrate a diabetes-related impairment of EDHF-mediated hyperpolarization in ZDF rat mesenteric arteries and indicate that this deficit is specific to SK_Ca.