dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. TheHANDgenes have a complementary left–right cardiac asymmetry of expression withdHANDpredominantly on the right side andeHANDon the left side of the looped heart tube. Here we show that althougheHANDis asymmetrically expressed along the anterior–posterior and dorsal–ventral embryonic axes, it is symmetrically expressed along the left–right axis at early stages of embryonic and cardiac development. After cardiac looping,dHANDandeHANDare expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left–right (LR) sidedness ofdHANDandeHANDexpression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however,dHANDexpression persists in the pulmonary ventricle andeHANDin the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. Previously we showed thatdHAND-null mice fail to form a right-sided pulmonary ventricle. Here mice homozygous for thedHANDandinvmutations are demonstrated to have only a right-sided ventricle which is morphologically a left (systemic) ventricle. These data suggest that theHANDgenes are involved in development of segments of the heart tube which give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals.