In a 2-lever operant chamber situation for food reinforcement, the effects of nicotine, arecoline and physostigmine on delayed discrimination responses in rats treated with ethylcholine mustard aziridinium ion (AF64A) were investigated. AF64A at 6 nmol, i.c.v., decreased the percentage of correct choices (% CR) at the delay time of 4 sec, and this effect depended on the delay time. On the other hand, AF64A at the same dose shortened the latency (LAT: the period from the end of the delay time to the initiation of lever-pressing responses) at the delay time of 16 sec, but this effect didn't depend on the delay time. Nicotine at 0.25 mg/kg, s.c., improved the decrease of % CR; and at 0.13-0.5 mg/kg, s.c., it shortened LAT at the delay time of 4 sec in rats treated with AF64A. Arecoline at 4 mg/kg, s.c., and physostigmine at 0.06-0.5 mg/kg, s.c., extended LAT, but both drugs showed no effects on % CR at the delay time of 4 sec in rats treated with AF64A. These results suggest that the present procedure might be useful for the evaluation of drug effects on memory in rats, and nicotine improved the delayed discrimination disruption induced by AF64A.