The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.