Purpose.To evaluate the relative contribution of leucine amino-peptidase and aminopeptidase III activities to the total amino-peptidase activity in bovine and human lenses underin vivopH conditions.Methods.Bovine and human lens extracts were fractionated on a Sephadex G-200 column at pH 6.9 and 8.5 and all the fractions were assayed with Leu-pNA and Arg-pNA as substrates atin vivoions pH (6.9) and optimum pH for leucine aminopeptidase, (8.5) The major peptidases were purified and their activities compared with that of LAP and AP III isolated from bovine lens. The ability of bovine and human lens extracts and purified bovine lens LAP and AP III to hydrolyze various peptide bonds in synthetic peptides, VHLPTVEK, bradykinin and Ile-Ser-bradykinin was determined by amino acid analysis of the reaction products.Results.Sephadex G-200 gel chromatography and assay of all the fractions at pH 6.9 showed that the elution volume for the predominant aminopeptidase present in bovine lens extract is the same as that of purified AP III from the same lenses. However, when the assays were done at pH 8.5, the major activity eluting from the Sephadex G-200 column was found in fractions having LAP. A similar study of human lens extracts at pH 6.9 and 8.5 showed one major peak with elution volume corresponding to that of purified bovine lens AP III. The human lens extracts displayed a very low level of LAP activity. The hydrolysis pattern of peptide substrates by AP III paralleled that of bovine and human lens extract at pH 6.9. The X-Pro bond resistant to LAP in peptide substrate, VHLTPVEK was hydrolyzed by AP III as well as lens extracts.Conclusions.Both bovine and human lenses have very low LAP activity compared to AP III activity atin vivopH 6.9. AP III, by its higher activity, broad specificity and its ability to cleave peptide bonds that are resistant to LAP, is likely to play a major role in lens during epithelial cell differentiation into fiber cells and complete hydrolysis of peptides generatedin vivo.