To assess the role of the aziridinium moiety for the cholinotoxicity of ethylcholine aziridinium ion (AF64A) we compared in vitro and in vivo effects of AF64A with those of various precursors as well as decomposition products of AF64A. In vitro, AF64A was the most effective irreversible inhibitor of high-affinity choline transport (HAChT) in hippocampal synaptosomes. The uncyclized precursor acetylethylcholine mustard and the acetylated form of AF64A were about 3 times less potent. Their potency, however, was reduced considerably when hydrolysis of the choline esters was prevented by physostigmine. Destruction of the aziridinium ring either by high pH (alcohol formation) or by thiosulfate (formation of Bunte salt) resulted in a loss of biological activity. This was also the case for the in vivo cholinotoxicity, as assessed by the decline in hippocampal concentration of acetylcholine (ACh) 7 days after intracerebroventricular (i.c.v.) infusion. The most pronounced reduction in ACh content was achieved after i.c.v. infusion of AF64A, whereas the precursor and the acetylated analog of AF64A induced a significant, but smaller reduction in the ACh content. These data indicate that the aziridinium ring of AF64A is essential for both the inhibition of HAChT in vitro and the cholinotoxicity in vivo. However, cyclization of the precursor compound as well as hydrolysis of acetylated AF64A also occur in tissue, leading to a partial activity of these compounds.