Endothelium-derived relaxing factors, such as nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) are thought to play an important role in vasodilation of penile arteries, and hence for erectile function. The present study investigated the mechanisms involved in flow-and acetylcholine-induced vasodilation in penile arteries, and whether acetylcholine- and flow-mediated vasodilation is altered in 18–20 week old Zucker diabetic fatty (ZDF) rats, a model of type-2 diabetes. Moreover, it was addressed whether enhanced myogenic tone may explain impaired flow-evoked vasodilation in arteries from ZDF rats. Penile dorsal arteries obtained from lean control and ZDF rats were suspended in a pressure myograph and flow and acetylcholine-evoked vasodilation was measured as changes in arterial diameter. Incubation with an inhibitor of NO synthase, asymmetric dimethylarginine (ADMA), and of cyclooxygenase, indomethacin, reduced acetylcholine, but not flow-evoked vasodilation in penile arteries, while both responses were abolished by endothelial cell removal. Iberiotoxin, a blocker of large-conductance Ca2⁺- activated K⁺ channels (BKCa), inhibited flow-evoked vasodilation. Flow-evoked vasodilation was reduced in arteries from ZDF rats in the absence, but not in the presence of indomethacin plus ADMA. In arteries from ZDF rats, myogenic tone increased by elevation of intraluminal pressure was less than in arteries from lean control rats. The present findings show that flow evokes endothelium- dependent EDHF-type vasodilation involving BKCa channels in penile arteries. Flow-evoked vasodilation is reduced and only of EDHF-type in penile arteries from type-2 diabetic rats suggesting modulation of this pathway may restore endothelial function and preserve erection in diabetes.