The choline analogue, ethylcholine mustard azirinium ion (AF64A), has been proposed as a selective neurotoxin that produces degeneration of central cholinergic neurons. However, the mechanisms of action and the specificity or non-specificity of this toxin are still undefined. In this study, we have investigated the effects of AF64A, in comparison with kainic acid, on cholinergic neurons of the mesopontine formation (pedunculopontine and laterodorsal tegmental nuclei), a neuronal population also expressing nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide. We used choline acetyltransferase immunohistochemistry as a marker of acetylcholine activity, and nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry as markers of nitric oxide synthase activity. Our results show that the injection of low doses of AF64A produces: (1) an area of cavitation in the injection site of pedunculopontine tegmental nucleus (local non-specific effect), and (2) a transient decrease in choline acetyltransferase immunoreactivity in choline acetyltransferase-nitric oxide synthase neurons in both the ipsilateral laterodorsal tegmental nucleus and the perilesional area of the pedunculopontine tegmental nucleus, while their morphology and nitric oxide synthase immunoreactivity remain unaltered (post-diffusion specific effect). These findings indicate that the loss of choline-related enzymatic activity is not necessarily associated with degeneration of cholinergic neurons, and that the recovery of choline acetyltransferase immunoreactivity may arise from neurons whose activity is diminished during the first postinjection weeks. Taking into account that AF64A is a suitable tool to develop a reversible model of neurological disorders related to cholinergic deficit, further efforts should be directed toward elimination of its local non-specific effect.