It is unclear why the concentration of testosterone increases in the testicular vein after hemicastration without corresponding alteration in gonadotropins. The present work was undertaken to examine whether the testosterone levels could be modified by denervation of the testis in adult rats. Both systemic and testicular blood samples were collected either immediately before or 6 and 24 hours after hemicastration from the rats two weeks after denervation of either inferior spermatic nerves (ISN) or ISN plus superior spermatic nerves (ISN-SSN). Increase of testicular testosterone induced by hemicastration was significantly (P<0.05) inhibited in these rats, as compared with the sham animals (at 6 and 24 hours, ISN vs sham: 16.00±3.35 vs 42.72±13.85 and 26.93±8.68 vs 71.16±13.30 whilst ISN-SSN vs sham: 31.63±7.92 vs 60.61±18.11 and 27.70±8.93 vs 93.92±19.73 ng/ml, respectively), whereas no significant change in LH was observed in all the experimental groups. FSH underwent no alteration in all the ISN denervation groups, but a significant elevation was observed in the ISN-SSN denervation groups (P<0.05) before hemicastration. Therefore, it appears that the change in FSH is not the cause of the inhibition of testosterone increase in the hemicastrated rats after testicular denervation and that ISN plays an active role in regulation of testosterone increase induced by hemicastration.