Orexins are hypothalamic neuropeptides primarily involved in the regulation of food intake and arousal states. In addition, a role for orexins as central neuroendocrine modulators of reproductive function has recently emerged. Prepro-orexin and orexin type-1 receptor mRNAs have been detected in the rat testis. This raises the possibility of additional peripheral actions of orexins in the control of reproductive axis, which remains so far unexplored. To analyze the biological effects and mechanisms of action of orexins in the male gonad, we evaluated testicular expression of orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R) mRNAs in different experimental settings and the effect of orexin-A on testicular testosterone (T) secretion. Persistent expression of OX(1)R mRNA was demonstrated in the rat testis throughout postnatal development. In contrast, OX(2)R transcript was not detected at any developmental stage. Expression of OX(1)R mRNA persisted after selective elimination of mature Leydig cells and was detected in isolated seminiferous tubules at defined stages of the seminiferous epithelial cycle. In addition, testicular OX(1)R mRNA expression appeared to be under hormonal regulation; it was reduced by long-term hypophysectomy and partially restored by FSH replacement, whereas down-regulation was observed after exposure to increasing doses of the ligand in vitro. Moreover, OX(1)R mRNA expression was sensitive to neonatal imprinting by estrogen. Finally, orexin-A, in a dose-dependent manner, significantly increased basal, but not human choriogonadotropin-stimulated, T secretion in vitro. A similar stimulatory effect was observed in vivo after intratesticular administration of orexin-A. In conclusion, our present results provide the first evidence for the regulated expression of OX(1)R mRNA and functional role of orexin-A in the rat testis. Overall, our data are suggestive of a novel site of action of orexins in the control of male reproductive axis.