The mammalian 'Dlx' gene family consists of six members which encode homeobox-containing proteins. These proteins are widely expressed during development; the major sites of expression include the forebrain, branchial arches, axial and appendicular skeleton, epidermis and placenta. Both Dlx5 and Dlx6 are regulators of chondrocyte differentiation during skeletogenesis, however, the mechanisms underlying this function are not known. To provide further insight into the role(s) of Dlx5 and Dlx6 in chondrocyte differentiation, a cell line that differentiates in a similar manner to 'in vivo' chondrocytes was used. Microscopic analysis of these cells following overexpression of either 'Dlx5' or 'Dlx6' revealed a lower cell number compared to controls. Condensations were not seen and no chondrogenic nodules were formed, in contrast to control cultures. Real-time PCR analysis indicated that 'Dlx6' overexpression significantly reduced the activation of 'Col2[alpha]1' and 'Sox9' expression but upregulated 'osteocalcin' expression. These results indicate that Dlx6 may induce an alternate differentiation pathway of ATDC5 cells.