Recently proposed procedures for in vitro generation of new cartilage may be difficult to perform in humans because so many chondrocytes are needed for tissue engineering. In this study the authors investigated new, efficient, low-cost techniques for the isolation and culture of chondrocytes from the ear cartilage of the rabbit. They performed a low-density monolayer culture with a low concentration (0.5%, 1%) of human platelet supernatant and observed cell proliferation (seeding efficiency, deoxyribonucleic acid synthesis), matrix synthesis (glycosaminoglycan synthesis), and the expression of type I and type II collagen (reverse transcriptase polymerase chain reaction). Seeding efficiency was increased in 1% of platelet supernatant-treated cultures by two to three times compared with untreated controls. One percent platelet supernatant had increased the incorporation of [3H]-thymidine by 1.9 to 2.5 times at 72 hours compared with controls. Glycosaminoglycan synthesis was increased in platelet supernatant-treated chondrocytes at 96 hours compared with controls. Chondrocytes treated with 1% platelet supernatant showed a decreased expression of the type II collagen gene. Supplementation with a high concentration (10%) of the platelet supernatant provided the conditions for in vitro chondrocyte mass formation. These results indicate that proliferation and matrix synthesis of auricular chondrocytes is stimulated by a low concentration of platelet supernatant. On the other hand, chondrocytes were immobilized by a high concentration of platelet supernatant. Platelet supernatant may be useful as an inexpensive autologous source of multiple growth factors to enhance chondrocyte proliferation, and also may play the role of scaffold for chondrocytes. Additional investigation is underway to generate culture conditions that promote the differentiation as well as the proliferation of chondrocytes.