Objective: Testosterone significantly improves hypogonadal-related erectile dysfunction(ED).However,the molecular mechanisms are poorly understood.The purpose of this study was to explore the effect and mechanism of testosterone in castrated rats.Methods: Forty male Sprague-Dawley rats were randomized to 4 groups(control,sham-operated,castration and castration-with-testosterone-replacement).After 2 months,reactive oxygen species(ROS)production was measured by dihydroethidium(DHE)staining.Erectile function was tested by recording intracavernosal pressure(ICP)and mean arterial blood pressure(MAP).Protein expression levels were examined by Western blot.Results: Castration reduced erectile function,and testosterone restored it.The concentrations of testosterone,cyclic guanosine mono-phosphate(cGMP)and cyclic adenosine monophosphate(cAMP)were lower in castrated rats than in controls,and testosterone restored these decreases(each P<0.05).The expression levels of cyclooxygenase-2(COX-2),prostacyclin synthase(PTGIS or PGIS),endothelial nitric oxide synthase(eNOS)and phospho-eNOS were reduced in castrated rats compared with controls.The expression levels were significantly elevated in rats treated with testosterone(each P<0.05).The expression levels of p40phox and p67phox were increased in castrated rats,and testosterone significantly reduced these increases(each P<0.05).ROS production was markedly enhanced in castrated rats,and testosterone administration reversed this effect(P<0.05).Conclusions: Testosterone can ameliorate ED after castration by reducing ROS production and increasing activity of the eNOS/cGMP and COX-2/PTGIS/cAMP signaling pathways.