Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.