The effects of the density of human mesenchymal stem cells (MSCs) on their differentioation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGF beta 3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05x10(4) to 0.9x10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05x10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05x10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.