A loss in the number of functional, sodium ion-dependent, high-affinity choline transport sites was observed in the cortex and hippocampus of mice given an intracerebroventricular injection of 65 nanomoles of AF64A (ethylcholine mustard aziridinium ion) 3 days earlier. Such an effect was not observed in the striatum. This effect of AF64A represents a long-term neurochemical deficit at cholinergic nerve terminals in some brain regions which can lead to a persistent deficiency in central cholinergic transmission. The AF64A-treated animal may thus be a model for certain psychiatric or neurological disorders that appear to involve central cholinergic hypofunction.