Cartilage damage is a major problem in osteoarthritis (OA). To gain insight into the pathogenesis of OA, we have analyzed the differential proteome of articular chondrocytes from these patients. Protein extracts were prepared from cultured chondrocytes from 6 patients with end-stage OA and 6 normal donors and were analyzed by 2D-DIGE. Differentially expressed proteins were identified by mass spectrometry (MS). Significant differential expression was observed for 27 proteins, with 14 underexpressed and 13 overexpressed chondrocyte OA proteins. Of special interest was the identification of destrin, cofilins, gelsolin, annexin A2, glycolytic enzymes, chaperones, cathepsin D, proteasome beta 9 subunit isoform 2 proprotein and proteasome activator hPA28 subunit beta. The altered expression of these proteins is consistent with events such as cytoskeleton binding, protein disruption, apoptosis, and glycolysis, demonstrating the ability of the 2D-DIGE/MS platform to identify proteins with altered expression in chondrocytes from patients with end-stage OA. The identification of these proteins may open new lines of research for this disease.