The Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na(+) channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.