We applied the scaffold-free culture method to chondrocytes and attempted to reconstitute articular cartilage grafts. Primary rat costal chondrocytes were immobilized into hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm(3) to induce the formation of cylindrical-shaped multicellular aggregates (organoids) and cultured for one month. The organoids were evaluated by histological and gene expression analyses. Chondrocytes formed cylindrical organoids in hollow fibers (HFs). Histochemical analysis revealed the accumulation of a cartilage extracellular matrix (collagen and proteoglycan) around cells in the lumen of HFs with culture time, forming a low-cellular-density tissue similar to native cartilage by day 28. Furthermore, in contrast to that in traditional monolayer culture, the organoid maintained the gene expression of the cartilage extracellular matrix (type II collagen, aggrecan) for one month of culture. In conclusion, our organoid formation method was effective in producing a cartilage-like tissue. This result suggests that the technique may be applicable to the development of an articular cartilage graft.