Exposure of articular cartilage to H2O2 in vitro inhibits proteoglycan synthesis in a fashion which parallels the inhibition which occurs in cartilage in animal models of acute inflammation. Our study shows that exposure to H2O2 also inhibits other chondrocyte functions, including total protein and DNA synthesis. Since these intracellular biosynthetic processes require adenosine triphosphate (ATP), the effect of exposure of H2O2 on chondrocyte ATP was measured. Exposure to H2O2 caused an immediate (less than 2 min) dose dependent decrease in cartilage ATP levels--found to be due to the oxidative inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH). We suggest that intrachondrocyte oxidant damage occurs through oxidation of the sensitive thiol (-SH) residue at the active center of G-3-PDH, with subsequent reduction in the rate of glycolytic ATP synthesis and the intracellular concentration of ATP which is required for DNA, protein, proteoglycan and hyaluronic acid synthesis.