The implication of myocardin and homeodomain only protein (HOP) in combinatorial molecular pathways that guide heart development and cardio-specific gene expression has recently been reported. However, expression of these genes in the failing heart has not yet been investigated. This study was designed to elaborate a molecular profile of myocardin and HOP expression in the failing ventricular myocardium through the use of both explanted human heart samples and heart biopsies from neonatal piglets with doxorubicin-induced cardiomyopathy (Dox-CM). Myocardin and HOP mRNA levels were estimated by both northern blot hybridization and semiquantitative RT-PCR in human ventricular preparations in end-stage failure due to dilated cardiomyopathy (DCM), as well as in nonfailing donor hearts. Similar experiments were performed with ventricular samples from normal and Dox-treated neonatal piglets. The gene expression of brain natriuretic peptide (BNP) was used as a molecular marker of myocardial damage and failure. The study revealed the following novel findings: (1) myocardin transcripts are detected in neonatal human and pig hearts at lower levels than in mature cardiac tissues, (2) the myocardin transcript pool is significantly augmented in the failing human and porcine myocardium as compared to that in nonfailing heart samples, (3) in the failing human myocardium, increased levels of myocardin mRNA are associated with a diminished HOP transcript content, and (4) the inverse proportion in cardiac myocardin/HOP mRNA pools observed in explanted human hearts is also traceable in normal human heart and aorta. A possible dual consequence of increased myocardin and decreased HOP expression levels on serum response factor-dependent cardiac-specific expression in the normal heart and at heart failure is discussed. Therefore, increased abundance of the myocardin mRNA pool is judged to be a novel CM-related feature which, alone or in association with decreased HOP transcript levels, can be responsible for dysregulation of myocardin-mediated gene expression in failing myocardium.