Indirect evidence suggests that ejaculation might activate endogenous opioid systems, which exert an inhibitory influence on male rat sexual behaviour. The objective of the present study was to search for putative long-term changes in the contents of immunoreactive (IR) Met-enkephalin (IR-Met), Leu-enkephalin (IR-Leu) and opioid octapeptide Met--Arg(6)--Gly(7)--Leu(8) (IR-Oct) in specific brain areas, after the execution of different amounts of sexual activity. Additionally, basal contents of these enkephalins were compared between sexually active (SA) and persistent sexually inactive (SI) rats. Immunoreactivity to enkephalins was determined by radioimmunoanalysis, in the frontal cortex, the hypothalamus and midbrain of SA and SI rats, as well as 24 or 48 h after males had one ejaculation or copulated to exhaustion. Twenty-four hours after sexual activity, there was a generalised increase in enkephalin contents that returned to control values at the 48 h measurement in all brain areas, but the hypothalamus, where IR-Met and IR-Oct remained elevated. No differences in the magnitude of the changes were found between rats that ejaculated once and sexually satiated males. IR-Oct concentration in the hypothalamus of SI rats appeared significantly higher than in SA animals, with no differences in IR-Met and IR-Leu. Results give direct evidence of the activation of endogenous opioid systems by male rat sexual activity. The occurrence of long lasting increases in the contents of IR-Met and IR-Oct in the hypothalamus of rats that copulated was detected. Finally, an intrinsically elevated octapeptide concentration in the hypothalamus of SI rats was found.