The aim of this research project was to investigate the possibility of developing the neurotoxic esterase (NTE) assay into a totally in vitro system to enable neurotoxicity assessment to be carried out rapidly on large numbers of compounds. The purified enzyme was to be immobilized on a solid matrix for use in a continuous-flow reactor. Occurrence of NTE was confirmed for brain, spinal cord, and peripheral nerve of the hen. The enzyme was absent from most other tissues, with the notable exception of spleen, spleen lymphocytes, and circulating blood lymphocytes. Chick brain NTE appears to be identical to hen brain NTE with respect to a variety of characterization parameters, and is therefore recommended as a tissue source for future work on NTE. In both tissues, the greatest subcellular enrichment is in the microsomal (P3) fraction. NTE may be solubilized from hen or chick brain microsomes with retention of inhibitor characteristics of the native enzyme. Solubilized NTE is most stable when stored at -18C in unbuffered Triton X-100 detergent solution. Immobilization of NTE was most successful using ionic binding to DEAE-Sephacel TM. However, this preparation was less stable than soluble enzyme stored under identical conditions.