Lens neutral proteinase is thought to exhibit primarily endopeptidase activity. We have identified a synthetic endopeptidase substrate which is hydrolyzed by the bovine lens neutral proteinase preparation. Among 11 fluoro- and chromogenic endopeptidase substrates, only carbobenzoxy-glycylglycyl-L-leucyl-p-nitroanilide is effectively hydrolyzed. The activity hydrolyzing this substrate co-elutes with neutral proteinase activity upon gel filtration and specifically attacks the leucyl-p-nitroaniline bond. Optimal hydrolysis of the synthetic substrate is at neutral pH and high temperature (53 degrees C), analogous to the alpha-crystallin protein substrate obtained from lens. The rate of hydrolysis of the synthetic substrate increased proportionally with temperature between 20 and 60 degrees C, in contrast to alpha-crystallin. The rate of hydrolysis was linear for at least 1 h at 37 degrees C and there was no evidence of enzyme activation at high temperature.