The substrate specificities of the esterases in rat pancreas and brain have been studied with organophosphorus compounds as selective esterase inhibitors. 2. The pancreas contains at least two enzymes with esterase activity which are relatively resistant to inhibition by diethyl p-nitrophenyl phosphate (E 600). The data obtained suggest that the E 600resistant activity towards salicyl butyrate is due to carboxypeptidase or an enzyme with similar properties, whereas the E 600-resistant activity towards ethyltyrosine is probably due to chymotrypsin. 3. The enzyme in pancreas identified previously as cholesterolesterase (Fodor, 1950; Myers et al. 1955 b) appears to be capable of hydrolysing a variety of esters including the ethyl esters of tyrosine, phenylalanine and leucine. The low activity towards these amino acid esters probably reflects the broad substrate specificity of this enzyme. 4. Three ali-esterases with distinct substratespecificity patterns could be distinguished in brain homogenates. These enzymes are distinct from the cholinesterases and from the cathepsin in brain, described by Krimsky & Racker (1949). 5. The possible physiological functions of these enzymes and the differentiation of esterases by means of selective inhibitors are discussed. The author is much indebted to Miss J. W. Tol and Miss M. H. T. de Jonge for invaluable technical assistance, and to Professor E. C. Slater for his suggestions concerning the preparation of this manuscript.