Chondrocytes isolated from cartilage tissue lose their chondrocytic phenotype during in vitro cell expansion, an event which has severely limited the advancement of tissue engineered cartilage and chondrocyte cell therapy. In an attempt to retard this loss of phenotype, we cultured costal chondrocytes in a supplementation of newborn calf serum(NCS) as opposed to the more commonly used fetal bovine serum(FBS). The costal chondrocytes cultured in the NCS medium maintained their chondrocytic phenotype longer than those cultured in the FBS medium, resulting in approximately five fold more cells expressing type II collagen and maintaining a round chondrocytic morphology after 3 weeks in culture. To evaluate their redifferentiation, the expanded chondrocytes were then seeded on a chitosan scaffold and cultured under NCS or FBS conditions. The differential effect of NCS on the maintenance of chondrocytic phenotype was also manifest in the high density three-dimensional culture, as evident by higher sustained expression of glucosaminoglycan than that seen in FBS medium. These results suggest that either FBS may act to stimulate the fibroblastic conversion of chondrocytes or that NCS may serve to retard it.