Aminopeptidase III activity was demonstrated in extracts from several different mammalian lenses by the hydrolysis of Arg-MCA at pH 6.0. No more than a two-fold difference was seen in overall specific activity. Sections of bovine lenses were removed from the periphery to the center and assayed. A sharp decline in activity was observed in the inner cortical region, and little or no activity was observed in the lens nucleus. This correlated with an increase in the presence of low molecular weight peptides as determined by SDS polyacrylamide gel electrophoresis. The properties of the aminopeptidase from human lens tissue were the same as those previously reported for the purified enzyme from bovine lens. The aminopeptidase activity of normal and cataractous lenses was compared using 4 different substrates. The cataractous lenses had significantly less total aminopeptidase activity. However, little difference in specific activity was observed based on soluble lens protein content. Similarly, electrophoretic separations of normal and cataractous soluble proteins showed little or no differences in the content of low molecular weight peptides. Therefore, this major human lens aminopeptidase remains functional in the cataractous state.