Myocyte enhancer factor (MEF2) are MADS box transcription fa:tors that play important roles in the regulation of myogenesis and morphogenesis of muscle cells. MEF2 proteins are activated by mechanical overload in the heart. In this study, we found the interaction of MEF2C with the regulatory protein Ki-1/57 using yeast two-hybrid system. This interaction was confirmed by GST-pull down assay in vitro and by co-immunoprecipitation in vivo. This interaction is also dependent on pressure overload in the heart. Co-imunoprecipitation assay with anti-MEF2 and anti-Ki-1/57 antibodies demonstrated a basal association between these proteins in the left ventricles of control rats. Pressure overload caused a reduction in this association. Ki-1/57 co-localizes with MEF2 in the nucleus of myocytes of control rats. However, after submitting the animals to pressure overload Ki-1/57 leaves the nucleus thereby decreasing this co-localization. Ki-1/57 also exerts an inhibitory effect upon MEF2C DNA binding activity. These results suggest that Ki-1/57 is a new interacting partner of MEF2 protein and may be involved in the regulation of MEF2 at the onset of hypertrophy. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.5791226152622