Background: Cell-based treatment approach using differentiated mesenchymal stem cells (MSCs) and mature chondrocytes has been considered as an advanced treatment for cartilage repair. We investigated the differentiated level of these two cell types that is crucial for their repair capacity for cartilage defect at a co-culture micro mass system. Methods: Passaged-2 MSCs isolated from the mouse bone marrow and the primary-cultured chondrocytes obtained from rat costal cartilage were mixed at different ratios including 1:1, 1:2, and 2:1, and cultivated in the micro mass culture systems (experimental groups). Both the MSCs and chondrocytes alone in micro mass cultures were considered as the controls. After 21 days, the cultures were sectioned and examined by toluidine blue staining. Furthermore, the cells at different groups were analyzed by semiquantitative reverse transcription-polymerase chain reaction using the specific primers designed to detect the expression of both mouse and rat cartilage-specific genes. Results: According to the toluidine blue staining, metachromatic stain appeared to be more intense at 1:2 ratios than the other groups. Based on semiquantitative analysis, all co-cultures possessed significantly more cartilage-specific gene expression than the controls (P<0.01). While mouse aggrecan and collagen II genes had significantly more expression at 1:2 ratio, rat collagen II gene was expressed in higher rate at co-culture with 2:1 ratio (P<0.01). Conclusion: Co-culture of MSCs with mature chondrocytes seemed to provide an appropriate microenvironment whereby the two cell types exhibit higher differentiated phenotype than when they were cultured alone, and sufficient to be used as the cellular material for repair of cartilage defects.