A dose of oxytocin (50 ng i.c.v.) that induces penile erection and yawning, increased the concentration of NO2- from 0.98 +/- 0.29 to 4.2 +/- 0.79 microM and of NO3- from 5.6 +/- 0.33 to 12.03 +/- 0.99 microM in the dialysate from the paraventricular nucleus of the hypothalamus of male rats, as measured by in vivo microdialysis. NO2- concentration was also increased by [Thr4, Gly7]-oxytocin (100 ng i.c.v. and oxytocin(8) (1 microgram i.c.v.) which also induced penile erection and yawning, but not by oxytocin(1-6) (1 microgram i.c.v.) or oxytocin (7-9) 1 microgram i.c.v.), which were unable to induce these behavioral responses. The oxytocin effect on NO2 concentration, penile erection and yawning was prevented by the oxytocin receptor antagonist. d(CH2)5,Tyr(Me)-Orn8-vasotocin (1 microgram i.e.v.) or by the nitric oxide synthase inhibitor, NG-nitro-1-arginine methyl ester (200 micrograms i.c.v.), but not by the dopamine receptor antagonist, haloperidol (0.5 mg/kg i.p.). The nitric oxide scavenger, hemoglobin (200 micrograms i.c.v.), prevented oxytocin-induced NO2- concentration increase, but was unable to prevent penile erection and yawning. Methylene blue (300 micrograms i.c.v.) an inhibitor of guanylate cyclase, was ineffective on oxytocin-induced NO2- concentration increase, but prevented the behavioral responses. The results suggest that oxytocin induces penile erection and yawning by increasing nitric oxide synthase activity in the cell bodies of oxytocinergic neurons projecting to extra-hypothalamic brain areas and mediating the behavioral responses.