The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region.