Heart development during embryogenesis involves mesodermal and neural crest-derived cell types. Mesodermal cells from a straight heart tube, which undergoes rightward looping followed by formation of atrial and ventricular chambers, while neural crest cells populate the developing aortic arches. Cell fate analyses have demonstrated that the straight heart tube is patterned into anterior to posterior segments that give rise to distinct compartments of the heart including the aortic sac, conotruncus (cardiac outflow tract), right ventricle, left ventricle, and atria, respectively. While several contractile protein genes are expressed in a chamber-specific fashion after cardiac looping, there have been no molecular markers for the early segments of the heart tube nor have regulatory factors that may control cell fates along the anteroposterior axis of the heart tube been identified. dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in mesodermal and neural crest-derived structures of the developing heart. We show that during mouse cardiogenesis, dHAND and eHAND were expressed in a complementary fashion and were restricted to segments of the heart tube fated to form the right and left ventricles, respectively. dHAND and eHAND represent the earliest cardiac chamber-specific transcription factors yet identified. Targeted gene deletion of dHAND in mouse embryos resulted in embryonic lethality at E10.5 from heart failure. dHAND-null mutants had only a single left-sided ventricle, failure of muscular trabeculation, and a grossly dilated aortic sac without evidence of branching aortic arch arteries. While most markers of cardiomyocyte differentiation were expressed in the mutants, the expression of the transcription factor, GATA-4, was down-regulated. The cardiac phenotype of dHAND mutant embryos demonstrates a requirement for dHAND in formation of the right ventricle and specific neural crest-derived compartments of the developing heart and reveals a novel cardiogenic subprogram for right ventricular development.