We have previously demonstrated the release of endogenous glutamate by activation of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors expressed by bone, while there is no information available on the possible functional expression of glutamatergic signaling molecules in cartilage to date. In rat costal chondrocytes cultured for 4 to 28 d, expression of mRNA was seen for several chondral marker genes including sox9, runt-related gene 2/core binding factor alpha-1 (Runx-2/Cbfa-1), type II collagen and aggrecan, but not for the adipocyte marker gene peroxisome proliferator-activated receptor gamma (PPARgamma). Expression of mRNA was drastically increased for Runx-2/Cbfa-1 during culturing from 7 to 14 d with a gradual increase thereafter up to 28 d, while a transient increase was seen in mRNA expression for both type-II collagen and sox-9 on 14 d and for aggrecan on 7 d respectively, in chondrocytes cultured for a period up to 28 d. Irrespective of the culture period up to 21 d, marked expression was seen by cultured chondrocytes with mRNA for GluR3 subunit of AMPA receptors, in addition to vesicular glutamate transporter-1 (VGLUT1) required for the condensation and subsequent exocytotic release of glutamate in the glutamatergic neurotransmission in the brain. Cultured rat costal chondrocytes underwent spontaneous release of endogenous glutamate, while an inhibitor of AMPA receptor desensitization significantly prolonged the duration of endogenous glutamate release stimulated by AMPA. These results suggest that endogenous glutamate could be released from intracellular vesicular constituents associated with VGLUT1 through activation of AMPA receptors expressed by cultured rat costal chondrocytes.