Hypoplastic right and left ventricles are among some of the most severe forms of congenital heart disease. It has been proposed that hypoplastic ventricles are the result of decreased growth stimulus secondary to diminished blood flow. We have previously reported that the basic helix-loop-helix transcription factor, dHAND, is specifically expressed in the segment of the heart tube fated to form the right ventricle. Targeted deletion of dHAND in mice resulted in a hypoplastic right ventricular chamber and other defects of the cardiac neural crest. We now propose a novel mechanism for dHAND's role in cardiac development. By intercrossing dHAND mutant mice with transgenic mice harboring a right ventricle-specific promoter (myosin light chain 2V) driving a lacZ reporter gene, we determined that a population of cardiomyocytes adopts a right ventriclular cell fate and differentiates appropriately. However, this population fails to expand during cardiac looping when the ventricular segments must increase in size. In vivo Brdu incorporation was normal in the mutant hearts indicating a normal proliferative rate. TUNEL assays revealed massive programmed cell death (apoptosis) in the segment of the heart tube fated to form the right ventricle well before any gross abnormalities or signs of heart failure in the mutants. Subtraction of mRNA pools between dHAND-null and wild-type embryonic hearts by representational difference analysis revealed that cytochrome c oxidase is down-regulated in dHAND-null hearts. In addition to its role in oxidative phosphorylation, release of cytochrome c from the mitochondrial membrane initiates the apoptotic pathway. Our data suggests a novel role for cytochrome c oxidase in preventing cytochrome c mediated apoptosis, possibly by retaining cytochrome c in the mitochondrial membrane. These data suggest that hypoplasia of an entire ventricle can occur with a single gene defect and is mediated by increased cell death of the hypoplastic chamber.