The venom of Crotalus durissus terrificus is reported to have analgesic activity and the administration of Crotoxin (Cro) to cancer patients is reported to reduce the consumption of analgesics. This study investigated the analgesia induced by Cro and the effects of atropine and naloxone on the antinociceptive activity of Cro in mice and rats. The results showed that Cro at 66.5, 44.3 and 29.5 μg/kg (ip) exhibited a dose-dependent analgesic action in mice using the hotplate and acetic acid writhing tests. Cro at 44.3 μg/kg (ip) had significant analgesic action in the rat tail-flick test. In the mouse acetic acid-writhing test, intracerebral ventricular administration of Cro 0.3 μg/kg produced marked analgesic effects. Microinjection of Cro (0.15 μg/kg) into the periaqueductal gray area also elicited a robust analgesic action in rat hotplate test. Atropine at 0.5 mg/kg (im) or 10 mg/kg (ip) or naloxone at 3 mg/kg (ip) failed to block the analgesic effects of Cro. These results suggest that Cro has analgesic effects mediated by an action on the central nervous system. The muscarinic and opioid receptors are not involved in the antinociceptive effects of Cro.