Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) decreased the sensitivity of rat cerebral cortical neurons to the excitant action of microiontophoretically applied acetylcholine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple barrel micropipettes tested on the same day. Acetylcholine sensitivity was determined by the E.T50 method. The E.T50 for 71 neurons in the caffeine-treated rats of 224.0 +/- 11.3 (S.E.M.) was significantly (P less than 0.001) greater than that of 65 neurons in the saline-treated control rats (153.8 +/- 6.9), indicating a reduction in the excitant action of acetylcholine on neurons which had been chronically exposed to caffeine. The level of spontaneous activity was also reduced in the caffeine-treated animals. A down-regulation of acetylcholine receptors is a possible cause for these effects.