An elevation in inorganic phosphate (P_i) concentration activates epiphyseal chondrocyte apoptosis. To determine the mechanism of apoptosis, tibial chondrocytes were treated with P_i, and nitrate/nitrite (NO/NO) levels were determined. P_i induced a threefold increase in the NO/NO concentration; inhibitors of nitric oxide (NO) synthase activity and P_i transport significantly reduced NO/NOlevels and prevented cell death. Furthermore, a dose-dependent increase in cell death was observed after exposure of chondrocytes toS-nitrosoglutathione. P_i increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protected chondrocytes from P_i-induced apoptosis. P_i caused a significant decrease in the mitochondrial membrane potential, while NO synthase inhibitors maintained mitochondrial function. While P_i caused thiol depletion, inhibition of P_i uptake or NO generation served to maintain glutathione levels. The results suggest that NO serves to mediate key metabolic events linked to P_i-dependent chondrocyte apoptosis.