Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca2+ accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.