The autologous chondrocyte transplantation technique has been introduced for the repair of articular cartilage defects. The advantage of transplanting chondrocytes cultured in suspension includes the in vitro expansion of cell numbers. However, the disadvantages include the potential leakage of cells from defects, dedifferentiation of cellular phenotype, and uneven distribution of cells. Transplantation of chondrocytes cultured in collagen gel resolves those problems. However, the expansion of cells in three-dimensional culture is more difficult than in monolayer culture, and for practical reasons only limited numbers of chondrocytes can be obtained from an unloaded area of the knee. To develop a method for the production of high-quality cultured grafts, we investigated the combination of monolayer culture for cell expansion and three-dimensional culture for maintenance of cell phenotype. Articular chondrocytes from rabbits were divided into four groups, exposed to various combinations of culture conditions, and cultured for a total of 3 weeks. Each group was evaluated histologically, biochemically, and biomechanically. Our findings showed that the combination of 2 weeks of monolayer culture followed by 1 week of three-dimensional culture resulted in the highest chondroitin sulfate levels, sufficient cell numbers, and adequate stiffness of the chondrocyte-collagen composites, giving optimal graft preparation.