A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterased showed that rat esterases were more sensitive than chicken to paraoxon inhibition at concentrations less than micromole and superimposable with chicken esterases at concentrations of 2.5-1000 micromole. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon concentration of 100 micromole (mipafox concentration: 0-1000 micromole) resulted in a mipatox 150 of 7.3 micromole for chicken brain NTE and 11.6 micromole for rat brain NTE. NTE(i.e., paraoxon-resistant, mipafox-sensitive esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for NTE from both species was similar showing broad, slightly alkaline optima from pH 7.9 to 8.6. (3H)Diisopropyl phosphorofluoridate (DFP)-labeled NTE from the brains of both species had an apparent mol wt of 160,000 measured by sodium dodecyl sulfate polyaacrylamide gel electrophoresis.