The relationship between total cholinesterase activity (TChE) and mortality in four butterfly species (great southern white [Ascia monuste], common buckeye [Junonia coenia], painted lady [Vanessa cardui], and julia butterflies [Dryas julia]) was investigated. Acute contact toxicity studies were conducted to evaluate the response (median lethal dose [LD50] and TChE) of the four species following exposure to the organophosphate insecticide naled. The LD50 for these butterflies ranged from 2.3 to 7.6 μg/g. The average level of TChE inhibition associated with significant mortality ranged from 26 to 67%, depending on the species. The lower bounds of normal TChE activity (2 standard deviations less than the average TChE for reference butterflies) ranged from 8.4 to 12.3 μM/min/g. As a percentage of the average reference TChE activity for the respective species, the lower bounds were similar to the inhibition levels associated with significant mortality, indicating there was little difference between the dose resulting in significant TChE inhibition and that resulting in mortality.