Androgens may exert cardiovascular protective actions by regulating the release and function of different vascular factors. In addition, testosterone (TES) and its 5-reduced metabolites, 5α- and 5β-dihydrotestosterone (5α- and 5β-DHT) induce vasorelaxant and hypotensive effects. Furthermore, hypertension has been reported to alter the release and function of the neurotransmitters nitric oxide (NO), calcitonin gene-related peptide (CGRP) and noradrenaline (NA). Since the mesenteric arteries possess a dense perivascular innervation and significantly regulate total peripheral vascular resistance, the objective of this study was to analyze the effect of TES, 5α- and 5β-DHT on the neurogenic release and vasomotor function of NO, CGRP and NA. For this purpose, the superior mesenteric artery from male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats was used to analyze: (i) the effect of androgens (10 nM, incubated for 30 min) on the neurogenic release of NO, CGRP and NA and (ii) the vasoconstrictor-response to NA and the vasodilator responses to the NO donor, sodium nitroprusside (SNP) and exogenous CGRP. The results showed that TES, 5α- or 5β-DHT did not modify the release of NO, CGRP or NA induced by electrical field stimulation (EFS) in the arteries of SHR; however, in the arteries of WKY rats androgens only caused an increase in EFS-induced NO release. Moreover, TES, and especially 5β-DHT, increased the vasodilator response induced by SNP and CGRP in the arteries of SHR. These findings could be contributing to the hypotensive/antihypertensive efficacy of 5β-DHT previously described in conscious SHR and WKY rats, pointing to 5β- DHT as a potential drug for the treatment of hypertension.