The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF, which are frequently inactivated in human cancer. Whereas p16 INK4a acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14 ARF were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14 ARF in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14 ARF in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14 ARF (Ad-p14 ARF). As expected, Ad-p14 ARF efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14 ARF also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53−/−). Similarly, adenovirus-mediated overexpression of p14 ARF induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax−/−). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14 ARF-triggered cell death. Infection with Ad-p14 ARF induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x L markedly inhibited p14 ARF-induced apoptosis. This may indicate that p14 ARF triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14 ARF-mediated apoptosis.