1. The effects of the intrahippocampal injection of ethylcholine mustard aziridinium ion (AF64A) on the following cholinergic markers were examined. 2. Bilateral injection of 10 nmol of AF64A into the dorsal hippocampus caused a loss of choline acetyltransferase (CAT) activity in the dorsal hippocampus, ventral hippocampus, cortex and striatum. 3. This treatment caused significant decreases in ACh contents in the dorsal hippocampus, ventral hippocampus and cortex, but not in the striatum. 4. The step-down latencies during the test trials of both 10 and 50 nmol AF64A-treated groups were significantly shorter than those of vehicle-treated groups. 5. None of the control animals stepped down to the large box where they had previously received a foot shock, but 40% of the 10 nmol and 60% of the 50 nmol AF64A-treated animals stepped down to the large box within 600 s. 6. These results indicate that AF64A treatment produces biochemical and functional deficits in the cholinergic neurons in the CNS.