1. The effect of atropine on the nociceptive system was examined in mice and rats by use of the hot-plate, writhing and tail-flick tests. 2. Atropine dose-dependently produced analgesia, no effect and hyperalgesia. Analgesia was observed in both species with doses ranging from 1 to 100 micrograms kg-1 while hyperalgesia was obtained with 5 mg kg-1. 3. Atropine antinociception was prevented by pirenzepine (0.1 microgram per mouse, i.c.v.), dicyclomine (10 mg kg-1, i.p.), atropine-methylbromide (0.5 microgram per mouse, i.c.v.) and hemicholinium-3 (1 microgram per mouse, i.c.v.). Naloxone (1 mg kg-1, i.p.), alpha-methyl-p-tyrosine (100 mg kg-1, s.c.) and reserpine (2 mg kg-1, i.p.) were ineffective. 4. The site of atropine analgesia is in the CNS since it exerts its antinociceptive effect also when injected i.c.v. (1-10 ng per mouse). Moreover drugs which do not cross the blood-brain barrier, such as hemicholinium-3, pirenzepine and atropine methylbromide, were unable to antagonize atropine analgesia if administered i.p. 5. Atropine also in vitro, showed a biphasic action on electrically-evoked guinea-pig ileum contractions. Concentrations between 10(-14) and 10(-12) M increased electrically and nicotine-evoked contractions but did not affect acetylcholine- and oxotremorine-evoked contractions. Concentrations above 10(-9) M inhibited both electrically- and drug (acetylcholine, nicotine and oxotremorine)-evoked contractions while they were ineffective on unstimulated ileum. 6. On the basis of the above findings, amplification of cholinergic transmission by very low doses of atropine is postulated, through a selective blockade of presynaptic muscarinic autoreceptors, as the likely mechanism of action. 7. Atropine antinociception, unlike oxotremorine antinociception, was obtained without any impairment of mouse rota-rod performance. 8. The antagonism by pirenzepine and dicyclomine of oxotremorine and atropine antinociception suggests that M1 muscarinic receptor subtypes are responsible for cholinergic analgesia.