This study assessed whether the in vivo production of nitric oxide (NO) in the penis is impaired in experimental diabetes and whether this phenomenon can be explained by abnormal levels of NO synthase isoenzymes and/or plasma androgens. Adult male Sprague-Dawley rats were injected with streptozotocin (STZ) (40 mg/kg, i.p.) or vehicle. One half of the STZ-treated animals received daily insulin replacement. Twelve weeks later, the animals were tested for mating behavior and erectile reflexes. They were then anesthetized with urethane (1 g/kg), and the NO levels in their corpora cavernosa were monitored electrochemically with porphyrin microsensors before and after electrostimulation of the cavernous nerve. The intracavernous pressure (ICP) was measured simultaneously. The diabetic animals had substantial impairment in the mating and erectile reflexes tests, decreased basal and stimulated NO levels in the corpora, and a reduced ICP response to cavernous nerve stimulation. Insulin replacement fully reversed the effects of diabetes on the mating reflexes, the basal NO signals, and the ICP responses to electrical field stimulation and partially restored the stimulated NO release. Neither diabetes nor diabetes with insulin treatment had significant effects on serum testosterone levels or NOS isoform (nNOS, eNOS, and iNOS) protein content in penile homogenates, indicating that the changes found in erectile function were independent of such variables. These results also suggest that the diabetes-induced reduction in corporeal NO levels could be mainly due to the lack of some essential cofactors for NOS activity rather than to changes in the amount of enzyme proteins.