Animal serum used for tissue engineering approaches has unacceptable risk for contamination with infectious agents. In this study, a cytokine-rich autologous serum (CRAS) system was developed. Canine auricular chondrocytes were cultured in medium supplemented with either fetal bovine serum (FBS) or autologous canine serum, alone or supplemented with basic fibroblast growth factor (b-FGF). Cell proliferative capacity was higher in the CRAS cultures than in those cultured in FBS, with greater expression of aggrecan and type II collagen in the b-FGF-supplemented CRAS group. The chondrocytes were seeded onto an ear-shaped biodegradable polymer (poly-L-lactide:epsilon-caprolactone, 50:50) and cultured in a Bioflow reactor for 1 week, using the 3 different culture media indicated above, and subsequently implanted into nude mice. The best outcome (cartilage gene expression and morphologic properties) was seen with tissue-engineered constructs precultured in the b-FGF-supplemented CRAS media. These findings indicate a clinically realizable approach for tissue engineering of cartilaginous structures.