The effects of cell density on the proliferation and chondroitin sulfate synthesis of chondrocytes embedded in Atelocollagen gel were examined. Chondrocytes of 21 10-week-old Japanese white rabbits isolated by collagenase digestion were embedded in Atelocollagen gel and cultured in Dulbecco's modified Eagles medium at cell densities of 2 x 105 cells/ml (105 group), 2 x 106 cells/ml (106 group), and 2 x 107 cells/ml (107 group) for 4 weeks. Chondrocytes in the 105 group gradually proliferated more than the other two groups. In contrast, most chondrocytes in the 107 group showed increased capability to produce chondroitin 6-sulfate. Cartilage-like tissue was produced from high-density cultures (107 cells/ml), although a decrease in cell number was seen. Even in three-dimensional cultures, the proliferation and chondroitin sulfate synthesis of chondrocytes were influenced by the cell density. These results are informative for the clinical application of chondrocyte transplantation in three-dimensional cultures for cartilage repair.