Neuron-enriched cultures prepared from several different rat brain regions were incubated with 10 microM or 30 microM monoethylcholine mustard aziridinium ion (AF64A) under conditions (1 h, 37 degrees C in Krebs Ringer buffer) that reduced acetylcholine (ACh) synthesis coupled to high-affinity choline uptake, without affecting choline acetyltransferase activity. Co-cultures of septum-hippocampus and cultures of striatum were similarly sensitive to the AF64A-induced inhibition of ACh synthesis. However, ACh-synthesis recovered more rapidly in the striatal cultures than in septal-hippocampal co-cultures after AF64A washout. In septal-hippocampal co-cultures, neither tunicamycin (20 micrograms/ml) nor cycloheximide (0.5 microgram/ml) had any effect on the basal synthesis of ACh or its recovery within 24 h following 10 microM AF64A washout. However, the recovery of ACh synthesis in these co-cultures after 30 microM AF64A-washout was blocked by either tunicamycin or cyclohexamide. Neither tunicamycin nor cyclohexamide interfered with ACh-synthesis recovery after washout of 30 microM AF64A in striatal cultures. These studies suggest that the turnover of high-affinity choline transporters can be modulated in a brain-region specific manner in intact primary neuronal cultures.