This report describes the properties of a neutral protease that was synthesized and secreted into medium by intact cartilaginous growth plate in tissue culture. Bovine cartilaginous growth plate was grown for seven days in tissue culture, during which time the chondrocytes remained viable and metabolically active as determined by quantitation of trypan-blue exclusion and incorporation of 3H-cytidine. Protease activity, assayed by viscometry using proteoglycan monomer from cartilage as a substrate, was absent on day 1 but was present at high levels on days 2 through 5. The protease activity did not require activation and was highest at neutral and alkaline pH. Protease activity was abolished by twenty-millimolar EDTA but was unaffected by pepstatin, iodoacetate, and soybean trypsin inhibitor. In contrast to the high levels of activity of neutral protease that were present in tissue cultures of the intact growth plate, no protease activity could be detected when chondrocytes from the cartilaginous growth plate were grown in cell culture, even after sonication of the cells or activation with aminophenyl mercuric acetate or trypsin. Since hypertrophic chondrocytes probably do not survive the disruption of tissue that is involved in establishing cell cultures, these observations suggest that neutral protease is probably released into the medium by the hypertrophic chondrocytes that are present in the cultures of cartilaginous growth-plate tissue. It appears that the organization of the growth plate in tissue culture, as well as the maturation of proliferating chondrocytes into hypertrophic chondrocytes in tissue culture, may be required for synthesis of the neutral protease and its extracellular secretion by hypertrophic chondrocytes.