The objective of this investigation was to distinguish which of the catalytic features of enzyme action is principally responsible for conferring the observed insensitivity of trout brain acetylcholinesterase (AChE; EC 220.127.116.11) to in vitro inhibition by organophosphates. The experimental design consisted of comparing the kinetic constants for the hydrolysis of a series of acylthiocholine substrates as well as the inhibition constants for a homologous series of dialkyl p-nitrophenyl phosphates among AChE from rats, hens, and trout. Chicken and rat brain AChE failed to distinguish between acetyl- and propionylthiocholine as inferred from the comparable Michaelis-Menten constants (Km), whereas trout brain AChE exhibited a much higher affinity for acetylthiocholine than for either of the two larger analogs. Diethyl p-nitrophenyl phosphate was the most potent inhibitor toward chicken and rat brain AChE, whereas the IC50 for the trout enzyme increased progressively between dimethyl and di-n-propyl p-nitrophenyl phosphate. The kinetic constants revealed that a significant determinant of inhibitor potency in the chicken and rat is steric exclusion as reflected by changes in the dissociation constant (Kd) which paralleled the changes in IC50 and ki. Conversely, Kd was 120- to 1450-fold higher and did not vary significantly for trout brain AChE. Instead, the phosphorylation rate constant (kp) for trout brain AChE decreased with progressive methylene substitutions. The kinetic data suggest that trout brain AChE not only possesses less steric tolerance, but also has a weaker nucleophile at the esteratic subsite, both of which may be important factors in conferring the observed insensitivity of trout to acute organophosphate intoxication.