Degradation of the proteoglycan matrix is considered an essential step in the process of calcification in the growth plate. This laboratory has just described the presence of a protease in human growth plate cartilage that degrades proteoglycan at neutral pH. We report here the isolation, partial purification, and characterization of these proteoglycan-degrading neutral proteases of bovine epiphyseal cartilage. It appears that there is more than a single enzyme active at neutral pH. These enzymes are of low molecular weight (below 30,000), poorly charged, and inhibited by metal chelating agents. Activity is best restored in the presence of zinc. This represents the first characterization of this important enzyme group.