Chromatin structure is a fundamental component of gene regulation, expression, and cellular differentiation. We have previously reported that the multifunctional coactivator p300 is a member of the Sox9 (Sry-type high mobility group box 9)-related transcriptional apparatus and activates Sox9-dependent transcription during chondrogenesis. However, the mechanism of synergy between Sox9 and p300 in chromatin-mediated transcription has not been elucidated. In the present study we investigated the activity of Sox9 and p300 on chromatinized templates in vitro. Recombinant Sox9 was shown to be associated with several transcriptional cofactors including p300. In vitro transcription assays revealed that p300 potentiated Sox9-dependent transcription on chromatinized DNA and, importantly, was associated with hyperacetylated histones. Consistent with these results, the histone deacetylase inhibitor trichostatin A stimulated the expression of Sox9-regulated cartilage matrix genes and induced histone acetylation around the enhancer region of the collagen alpha1 (II) gene in chondrocytes. These findings suggest that Sox9 interacts with chromatin and activates transcription via regulation of chromatin modification.