To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41 degrees C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37 degrees C were higher than the corresponding values at 32 degrees C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37 degrees C compared to those at 32 degrees C, whereas they were considerably inhibited at 41 degrees C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. (c) 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:633-639, 2015.