2,5-Hexanedione is a n-hexane metabolite with neurotoxic properties. We have previously demonstrated that acute administration of 2,5-hexanedione causes analgesia in the tail-flick test in rats. In the present investigation, we examined the possible involvement of a cholinergic component in the 2,5-hexanedione-induced antinociception, since literature data indicate that this hexacarbon compound may act as a competitive inhibitor of acetylcholinesterase and that cholinesterase inhibitors are analgesic to rodents. Rats were treated with saline or with 5 or 25 mg/kg atropine (intraperitoneally) 10 min. before the injection of vehicle or 800 mg/kg 2,5-hexanedione (intraperitoneally). 2,5-Hexanedione caused a significant increase in tail-flick latencies at 10, 30, 60 and 90 min. after hexacarbon injection. Atropine (5 or 25 mg/kg) partially reversed the analgesia caused by 2,5-hexanedione at 60 and 90 min. When effects of 2,5-hexanedione on brain acetylcholinesterase was assessed in vitro, the results demonstrated that a competitive component is involved in enzyme inhibition. Taken together, these data support the involvement of a cholinergic (muscarinic) component in 2,5-hexanedione-induced analgesia.