The most recently discovered collagen gene, COL27A1, codes for type XXVII collagen. The COL27A1 gene is strongly expressed in developing cartilage and weakly expressed in many other tissue types. The present study was undertaken to identify transcriptional regulatory mechanisms that govern the expression of COL27A1 in cartilage, and in particular to determine whether SOX9, a key regulator of chondrogenesis, could activate COL27A1. The first intron of COL27A1 was examined to identify sites with homology to the Sox consensus sequence (A)/(T)(A)/(T)CAA(A)/(T)G. Three 50-bp regions that contained paired Sox sites arranged in opposite orientation to each other and separated by 3 or 4 bp were targeted for further analysis. The elements were tested by transient transfection of reporter plasmids, and two of the three elements showed enhancer activity in chondrocytic cells. The same two elements bound SOX9 in electrophoretic mobility shift assays (EMSA). They were not transcriptionally active in fibroblasts, but cotransfection with a SOX9 expression plasmid resulted in activation. The independent mutation of either Sox site in a pair prevented SOX9 binding to the enhancers in EMSA experiments, indicating that SOX9 binds these enhancers only as a dimer. Mutation of either site in a pair also abolished enhancer activity in chondrocytes, indicating that dimeric binding of SOX9 is required for transcriptional activation of the two new enhancers. In summary, these results suggest that SOX9 may play an important role in the transcriptional activation of the newest collagen gene, COL27A1.