Intracerebroventricular (i.c.v.) administration to mice of the venom tetradecapeptide mastoparan (MP), which binds certain G transducer proteins, resulted in a long-lasting reduction of the supraspinal antinociception induced by opioids and clonidine, the alpha-2 adrenoceptor agonist. The alpha N-acetyl derivative of beta-endorphin-(1-31) (NAC-beta-END; 1 pmol, i.c.v. per mouse) injected 1 hr before an equimolar dose of MP lessened the antagonist activity of the venom peptide on the antinociception induced by D-pen2,5-enkephalin, [D-Ala2]deltorphin II, D-Ala2-N-MePhe4-Gly-ol5-enkephalin and clonidine when evaluated 24 hr later with the tail-flick test. The impairment exerted by MP on these analgesic substances was not altered when NAC-beta-END was administered after the venom peptide. Therefore, the antinociceptive effect depends on which peptide was injected first. The protective activity of NAC-beta-END against MP antagonism of opioid/clonidine antinociception was dose related and exerted at attomolar and femtomolar doses. This finding strengthens the idea of a neural substrate (possibly G proteins) distinct from the opioid receptor enacted by NAC-beta-END to modulate opioid/clonidine antinociception. This research shows that a series of substances, neuropeptides and venoms dissimilarly influence the functional state of G proteins, thus altering the agonist-activated transduction cascade.