dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Here we show that although eHAND is 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, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. Previously we showed that dHAND-null mice fail to form a right-sided pulmonary ventricle. Here mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle which is morphologically a left (systemic) ventricle. These data suggest that the HAND genes 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.