Hypospadias is usually associated with androgen insufficiency. As androgens regulate Ca 2+ influx, Nox activity and redox signalling, we sought to ascertain where these may be implicated in vascular dysfunction seen in boys with hypospadias. Peripheral arteries dissected from excess foreskin tissue from hypospadias (cases) or circumcision (controls) were used. Vascular function was assessed by wire myography.VSMCs were cultured and ROS production was measured by amplex red and chemiluminescence. mRNA expression was measured by qPCR and Ca 2+ influx by fluorescence microscopy. 20 boys with hypospadias and 29 age-matched controls were enrolled in this study (median age 1.9 (range 1.3, 12.2) years). Arteries from cases demonstrated increased constriction to U46619 vs controls (Emax %KCl: 175.6 vs 66.3 p<0.05). Expression of Nox5 was increased in cases (2.6 fold,p<0.05), while a decrease in Nox4 expression was observed (9.5 fold, p<0.05). VSMC superoxide anion (5.3 fold) production and H 2 0 2 (3.3 fold) levels were increased in cases compared to controls (p<0.05), an effect inhibited by melittin. Melittin, a Nox5 inhibitor, inhibited contraction in arteries from boys with hypospadias (Emax %KCl: 137.9 vs 62.9, p<0.05). There was no difference in mRNA expression of androgen receptors ( AR and GPRC6A) between cases and controls but cases had higher expression of the oestrogen receptors, ESR1 (2.7 fold), ESR2 (2.6 fold) and GPR30 (2.9 fold) (p<0.05).Boys with hypospadias demonstrated increased CACNA1C (3.9 fold), SERCA (3.1 fold), TRPM2 (2.0 fold) and Ryr1 (3.5 fold) mRNA expression (p<0.05).There was no basal difference between Ca 2+ influx between controls and boys with hypospadias.Testosterone and oestradiol stimulation reduced expression of Ca 2+ channels (p<0.05). Moreover, testosterone (p=0.04) and oestradiol (p=0.02) reduced Ca 2+ influx in boys with hypospadias. Vascular dysfunction observed in small arteries from boys with hypospadias may be due to Nox5 activation and altered Ca 2+ regulation. These data suggest a role for androgens in the regulation of vascular Nox5-Ca 2+ signaling, which when perturbed may predispose to cardiovascular risk.