The comparison is between a pancreatic and a bacterial serine protease with similar specificities, similar sequences around their catalytically functional serine and histidine residues, and with certain notable similarities in amino acid composition.The two enzymes match one another in the following respects, (a) kc depends on an ionization with a pKa of approximately 6.7 in H2O and 7.2 in 2H2O; kc/Km is reduced by 50% when H2O is replaced by 2H2O; the data are consistent with general basic catalysis by a single, unprotonated histidine residue. (b) Km is independent of pH from pH 5 to 10.5; optical rotation is independent of pH over the same range. (c) Neither enzyme is inhibited by the chloromethyl ketones derived from N-tosylglycine and N-tosyl-L-valine or by the bromomethyl ketone derived from N-tosyl-L-valine.Both enzymes have elastolytic activity and both have lytic activity towards Arthrobacter globiformis cells, but elastase has the higher elastolytic activity and the α-enzyme the higher bacteriolytic activity. The α-enzyme is more thermostable at pH 8.0.A systematic comparison was made of their esterase activities as influenced by the side chains, the N-acyl substituents, and the alcoholic groups of amino acid esters. Esters of L-alanine were the best substrates for both enzymes; esters of L-valine were moderately good substrates for the α-enzyme but not for elastase; esters of glycine, L-leucine and L-isoleucine, and D-alanine were very poor substrates for both enzymes. Both enzymes are very sensitive to the nature of the N-acyl substituent and show appreciable differences in their response to various substitutions. The alcoholic group is a less critical determinant of activity but the values of kc are not independent of the nature of the group.A reaction mechanism is proposed in which the formation of an acyl intermediate is not obligatory but is a limiting case of a general mechanism.