The iris is innervated by both cholinergic parasympathetic, and adrenergic sympathetic branches of the autonomic nervous system. This innervation represents a simple and anatomically well-defined system to evaluate the effects of chemical compounds on cholinergic and adrenergic neurons. AF64A (acetyl ethylcholine aziridinium) is a known cholinotoxin in the brain and, in these experiments using the iris system, we evaluated its in vivo effect on cholinergic enzyme activity, pupillary size, and catecholamine neurotransmitter levels. We found in this system that AF64A reduces the activity of choline acetyltransferase (ChAT) but not acetylcholinesterase (AChE). AF64A is selective for cholinergic neurons, since norepinephrine and dopamine levels were unaffected.