The transcription factor Sox9 is capable of enhancing type II collagen gene expression and may play a crucial role in chondrogenesis. To determine whether Sox9 is an inducer of the chondrocyte phenotype, we investigated the role of Sox9 in transcription of another cartilage gene encoding the cartilage-derived retinoic acid-sensitive protein (CD-RAP). CD-RAP is specifically expressed during chondrogenesis. We show here that Sox9 protein is able to bind to a SOX consensus sequence in the CD-RAP promoter. Mutation of the SOX motif led to decreased transcription of a CD-RAP promoter construct in chondrocytes. Overexpression of SOX9 resulted in a dose- dependent increased activity of CD-RAP promoter-driven reporter gene in both chondrocytes and nonchondrogenic cells. A truncated SOX9, which contains a binding domain but no trans-activation function, inhibited CD-RAP promoter activity. Overexpression of SOX9 increased the level of endogenous CD-RAP mRNA in chondrocytes, but was unable to induce endogenous gene expression in 10T1/2 mesenchymal cells or BALB/c-3T3 fibroblasts. These results suggest that Sox9 is a general transcriptional regulator of cartilage-specific genes. However, Sox9 does not appear to be able to induce the chondrocyte phenotype in nonchondrogenic cells, implying that other factors are involved in chondrogenesis.