Acetylcholinesterase (AChE) preparations from the muscle of the fathead minnow (Pimephales promelas Rafinesque) were treated (in vitro) with 74 chemicals of various classes, many of which are environmental contaminants, to determine their effect upon enzyme activity. A highly inhibitory effect was found with several carbamates, one organo-oxy-phosphate, the arsenite ion, and certain heavy metal cations. Intermediate inhibition was found with the arsenate ion, other metal cations, organometals, certain neuroactive agents, organophosphates, and one organochloride pesticide. Several organochloride pesticides, alkaline and alkaline-earth cations, a variety of anions, several drugs, and other chemicals had little inhibitory effect at the highest concentration used (0.01 M). The effect of chemicals in mixtures of known composition was approximately additive. None of the chemicals tested caused detectable enzyme activation.