The retinoblastoma protein (Rb) controls cellular proliferation and suppresses tumor formation through its effects upon E2F transcriptional regulation of the cell cycle. Unexpectedly, however, in proliferating human cells, Rb was present at the promoters of eight of eight E2F-regulated apoptotic genes tested, but zero of six E2F-regulated cell cycle genes tested. Binding of apoptotic gene promoters by Rb was constitutive, and inhibition of Rb in human cells by E2Fdb or E1A expression resulted in induction of these apoptotic genes and efficient cell death. E1A induced apoptosis much more efficiently in human fibroblasts than in mouse fibroblasts, suggesting a difference in susceptibility to loss of Rb function between human cells and mouse cells. Abrogation of Rb function in mouse cells did not induce expression of these apoptotic genes. Underlying this species difference in susceptibility to apoptosis following loss of Rb function was the absence of Rb on apoptotic gene promoters in mouse cells. Rb protein levels were 20-35-fold higher in primary human cells than in primary mouse cells. The constitutive repression of a multitude of apoptotic genes by Rb in human cells but not in mouse cells may provide a partial explanation for the well-known difference between human and mouse cells in transformation and tumorigenic potential.