In the adult mongrel dog, In vivo injection of chymopapain into the intervertebral disc resulted, at two weeks, in disc space narrowing. However, [35S]sulfate labeling and proteoglycan characterization demonstrate that the nucleus retains the ability to synthesize proteoglycans, although they were degraded rapidly by residual proteolytic activity. Three months following chymopapain treatment, the intervertebral dog disc shows that an increase in disc height, return of nuclear material, and proteoglycan aggregate is present. At six months following chymopapain treatment, proteoglycans of similar characteristics to normal canine intervertebral disc are identified with a glucosamine/galactosamine ratio approaching normal values. Biomechanically, the short-term (30–120 minutes) In vitro effects of chymopapain appear to be the same as the carrier causing increased disc height, stiffness values, and creep rates. In the in vivo study, after three weeks, chymopapain-injected discs had significant reductions in disc height and compressive stiffness, but the creep rate was increased substantially. However, at three months postinjection, these biomechanical properties began to reverse and approached those of the uninjected controls. The observations reported in this study suggest that chymopapain has a profound but reversible effect on normal canine intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with loss of proteoglycan content, structure, and biomechanical properties. The restoration of normal disc height following chymopapain injection is explained by reconstitution of normal intervertebral disc. EDTA and cysteine used alone have no discernable in vivo enzymatic effect on intervertebral disc proteoglycan biochemistry. Chemonucleolysis with chymopapain would appear less likely to alter permanently proteoglycan biochemistry and the biomechanical properties of the disc than surgical excision in experimental animals.