The E2F transcription factors are key downstream targets of the retinoblastoma protein tumor suppressor. They are known to regulate the expression of genes that control fundamental biological processes including cellular proliferation, apoptosis and differentiation. However, considerable questions remain about the precise roles of the individual E2F family members. This study shows that E2F3 is essential for normal cardiac development. E2F3-loss impairs the proliferative capacity of the embryonic myocardium and most E2f3(-/-) mice die in utero or perinatally with hypoplastic ventricular walls and/or severe atrial and ventricular septal defects. A small fraction of the E2f3(-/-) neonates have hearts that appear grossly normal and they initially survive. However, these animals display ultrastructural defects in the cardiac muscle and ultimately die as a result of congestive heart failure. These data demonstrate a clear role for E2F3 in myocardial and cardiac function during both development and adulthood.