Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of chromatin, histone modification, and DNA methylation, strictly controls the pattern of gene expression and silencing. Recent studies have revealed that histone acetylation plays a crucial role in relaxing chromatin structure for initiation of transcription. Crosstalk between DNA-binding transcription factors and histone acetyltransferases (HATs) serves as a key mechanism for regulating gene expression and developmental processes. However, the precise roles of multicomponent transcriptional complexes have not been fully elucidated because of technical difficulties in using in vitro experimental systems. Previously we demonstrated that the DNA-binding transcription factor Sox9, HAT coactivator p300, and other regulatory factors (Smad3/4) cooperatively activate Sox9-dependent transcription on chromatin. Here, we describe an experimental approach to investigate the function of each component on reconstructed chromatin in vitro. Our methods offer a useful system for analyzing the additional effect of a third component in a transcriptional complex on chromatin structure.