Proteolytic enzymes are known to appear in tears only following trauma to the cornea. Aminopeptidases, enzymes involved in the final degradation of proteins to free amino acids, were measured and partially characterized for substrate specificity in normal human and animal tears. In all species tested, aminopeptidase activity was greatest for methionine β-naphthylamide of the various synthetic analogs of N-terminal L-amino acids. The substrate specificities for aminopeptidase(s) in tears for 11 different mammals were very similar. In children aminopeptidase activity decreased with increasing age and was unaffected by nutritional status.