The type X collagen gene (Col10a1) is a specific molecular marker of hypertrophic chondrocytes during endochondral bone formation. Mutations in human COL10A1 and altered chondrocyte hypertrophy have been associated with multiple skeletal disorders. However, until recently, the cis-enhancer element that specifies Col10a1 expression in hypertrophic chondrocytes in vivo has remained unidentified. Previously, we and others have shown that the Col10a1 distal promoter (−4.4 to −3.8 kb) may harbor a critical enhancer that mediates its tissue specificity in transgenic mice studies. Here, we report further localization of the cis-enhancer element within this Col10a1 distal promoter by using a similar transgenic mouse approach. We identify a 150-bp Col10a1 promoter element (−4296 to −4147 bp) that is sufficient to direct its tissue-specific expression in vivo. In silico analysis identified several putative transcription factor binding sites including two potential activator protein-1 (AP-1) sites within its 5′- and 3′-ends (−4276 to −4243 and −4166 to −4152 bp), respectively. Interestingly, transgenic mice using a reporter construct deleted for these two AP-1 elements still showed tissue-specific reporter activity. EMSAs using oligonucleotide probes derived from this region and MCT cell nuclear extracts identified DNA/protein complexes that were enriched from cells stimulated to hypertrophy. Moreover, these elements mediated increased reporter activity on transfection into MCT cells. These data define a 90-bp cis-enhancer required for tissue-specific Col10a1 expression in vivo and putative DNA/protein complexes that contribute to the regulation of chondrocyte hypertrophy. This work will enable us to identify candidate transcription factors essential both for skeletal development and for the pathogenesis of skeletal disorders.