The transcription factor SOX9 plays a critical role in chondrogenesis as well as in sex determination. Previous work has suggested that SOX9 functions as a DNA-dependent dimer when it activates genes involved in chondrogenesis, but functions as a monomer to activate genes involved in sex determination. We present evidence herein for a third binding configuration through which SOX9 can activate transcription. We have identified four separate SOX consensus sequences in a COL9A1 collagen gene enhancer. The sites are arranged as two pairs, and each pair is similar to previously discovered dimeric SOX9 binding sites. Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites. The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression. These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.