Structure-activity relationships reveal that the two principal determinants of anticholinesterase activity for various organophosphorus insecticides are steric hindrance and the electrophilic strength of the phosphorus atom. The objective of the present investigation was to distinguish between the molecular properties governing species-related differences in organophosphorus sensitivity by comparing the physical-chemical relationships for the inhibition of brain acetylcholinesterase isolated from rats, chickens, or rainbow trout. A homologous series of five dialkyl p-nitrophenyl phosphates consisting of methyl through n-butyl and isopropyl were synthesized and characterized both chemically and biologically. Structure-activity correlations revealed that whereas steric hindrance is the principal factor governing inhibitory potency for rats and hens, the electrophilicity of the phosphorus atom is the principal determinant of anticholinesterase activity in trout. The inductive effect of successive methylene substitutions on the phosphoryl group is reflected by 13C and 31P NMR spectroscopy which correlates with anticholinesterase activity in trout, but not in rats or hens. The results provide the first indication for species-related differences in the molecular regulation of anticholinesterase activity, suggesting that the trout enzyme possesses a relatively weak nucleophilic center within a dimensionally restricted esteratic subsite. Species-specific distinctions in the molecular properties governing anticholinesterase activity provide novel design strategies for improving the selectivity of toxic organophosphorus insecticides.