BACKGROUND: In end-stage osteoarthritis (OA), osteochondral defects reach the subchondral bone and cartilage tissue of sufficient thickness is required to compensate for the defects. Adipose-derived mesenchymal stem/stromal cells (ADSCs), which are abundant in the body, have the potential to differentiate into cartilage and may be a useful cell source for cartilage regeneration. If it is possible to fabricate ADSC-derived cartilage constructs that can cover the damaged area, this could lead to the development of a new regenerative therapy for OA that could replace the currently available treatments. We therefore sought to produce cartilage constructs with suitable thickness and biological properties, similar to native cartilage, using the bio-three-dimensional (3D) printer. We also investigated the culture protocol to ensure that the constructs were fully mature even at the internal site. METHODS: ADSCs were isolated from three rats and expanded to create cartilage spheroids. The spheroids were arranged into patches using a Kenzan bio-3D printer to create scaffold-free, cell-only cartilage constructs. Basic fibroblast growth factor (bFGF) was added during expansion culture and varying concentrations of bone morphogenetic protein2 (BMP2) were supplemented during chondrogenic differentiation. The levels of glycosaminoglycans (GAG) in the spheroids and constructs were measured. The histology of the spheroids and constructs and the compressive strength of the constructs were evaluated. RESULTS: The amount of GAG in the cartilage spheroids was higher in the bFGF and high-BMP2 concentration groups than in the non-supplemented and low-BMP2 concentration groups. Chondrocytes were abundant in the spheroids and constructs, and the extracellular matrix was homogeneously positive for safranin O staining and type II collagen immunostaining. The strength of cartilage constructs was consistent with that of the native cartilage (compressive strength 4.2 ± 1.5 MPa, n = 12). CONCLUSION: By optimizing the cell culture conditions, inducing chondrogenic differentiation, and bio-3D printing, we successfully fabricated fully mature cartilage constructs with mechanical and histological properties similar to those of articular cartilage.