Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo . By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b, as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4. However, the precise functional relationships among p300, GATA4, and Cdk9 remain unknown. Methods and Results: A series of GST pull-down assays revealed that the N-terminal of Cdk9 bound to the N-terminal zinc finger domain of GATA4 and C/H-3 domain of p300, respectively. By chromatin immunoprecipitation and DNA pull-down assay, we showed that GATA4 recruited Cdk9 onto GATA elements within the endothelin-1 promoter. Immuno-precipitation followed by Western blotting demonstrated that intact p300 induced not only the acetylation of GATA4, but also the interaction between GATA4 and Cdk9. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in the regulation of Cdk9 kinase activity. All of these effects were inhibited by the co-expression of a dominant-negative form (DN-) of p300. Conversely, Cdk9 induced not only the hyperphos-phorylation of RNA Pol II, but also the phosphorylation of p300. Notably, Cdk9 induced the acetylation and DNA binding of GATA4. However, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, was unable to achieve these, suggesting the requirement of Cdk9 kinase activity for p300 HAT activity. Finally, a Cdk9 kinase inhibitor, DRB, inhibited phenylephrine-induced hypertrophic responses as well as the acetylation of GATA4 in cardiomyocytes. Conclusion: These findings demonstrate that Cdk9 is required for the phosphorylation of p300 and its HAT activity, and forms a functional complex with p300/GATA4 during cardiomyocyte hypertrophy.