The objective of the present study was to assess the effect of endogenous male sex hormones on the activity of protein kinase C (PKC), as well as the regulatory effect of this kinase on the neuronal nitric oxide (NO) release induced by electrical field stimulation (EFS; 200 mA; 0.3 ms; 1–16 Hz). For this purpose, superior mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. PKC activity was greater in arteries from orchidectomized than control rats. Basal and EFS-induced NO release was similar in arteries from both groups despite the lower nNOS expression in arteries from orchidectomized rats. Phorbol 12,13-dibutyrate (PDBu), a PKC activator, EFS-induced NO release was higher in arteries from control compared to orchidectomized rats. Calphostin C, a non-selective PKC inhibitor, or Gö6976, a PKC inhibitor partially selective for conventional isoforms,abolished the EFS-induced NO release in arteries from control animals, while it was decreased in arteries from orchidectomized animals. The PKCζ pseudosubstrate inhibitor decreased EFS-induced NO release equally in both groups. The NO synthase (NOS) inhibitor Nω-nitro- L-arginine-methyl ester (L-NAME) enhanced the EFS-elicited contractions in arteries from both groups. Calphostin C increased the contractions elicited by EFS in arteries from control and orchidectomized rats. This increase was further enhanced by calphostin C plus L-NAME only in orchidectomized rats. PDBu reduced EFS-induced contraction in arteries from controls but did not affect it in orchidectomized rats. The further addition of L-NAME increased the responses in both types of arteries. These results show that PKC activity is enhanced in mesenteric arteries from orchidectomized rats, which may be the responsible for the greater nNOS activity in these arteries. Conventional and atypical PKCζ isoforms positively regulate nNOS activity in arteries from both control and orchidectomized rats, but the contribution of conventional PKC isoforms to enhanced nNOS activity seems to be greater in arteries from orchidectomized rats.