The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis. Retroviral SOX9 transduction of passaged chondrocytes increased the endogenous rate of GAG synthesis and the total capacity for GAG synthesis assessed in monolayer culture with beta-xyloside. Both the endogenous rate and the total capacity of GAG biosynthesis were increased further in chondrogenic cell aggregate cultures. The GAG synthesized was predominantly CS and the hydrodynamic size of the newly synthesized chains was unchanged by SOX9 transduction. Aggrecan gene expression was increased in the SOX9-transduced chondrocytes and increased further in chondrogenic culture, but no comparable effects were found in SOX9 transduced dermal fibroblasts. However, the expression of CS glycosyltransferase and sulfotransferase genes in chondrocytes was unaffected by SOX9 transduction. Therefore SOX9 transduction in chondrocytes increased their CS synthetic capacity, but this was not accompanied by changes in the transcription of the CS biosynthetic enzymes and must occur by indirect regulation of enzyme activity through control of enzyme protein translation or enzyme organization.