The INK4A locus is often inactivated in human cancer. INK4A encodes for p14ARF and p16INK4A that inhibit growth through p53 and pRb, respectively. We used RNA interference vectors in transformation assays of human primary cells to analyze tumor-suppressive functions. We first show that a concerted inactivation of pRb and p53 is required for transformation. We then demonstrate that loss of p14ARF enhances growth in a p53-dependent manner but has little tumorigenic effect. In contrast, suppression of p16INK4A expression does not affect cellular proliferation but synergizes with p53 loss to accelerate growth and cause transformation. Our results delineate the functions of the human INK4A genes in normal and tumorigenic growth.