We have analyzed the ability of an adenoviral vector encoding the exon 1beta region of the p14(ARF) tumor suppressor (ARF) to suppress the growth and viability of an array of tumor cell lines of various origins and varying p53 and Rb status, in order to establish the clinical potential of ARF. An important activity of ARF is regulation of p53 stability and function through binding to the mdm2 protein. By sequestering mdm2, ARF may promote growth suppression through the Rb pathway as well because mdm2 can bind to Rb and attenuate its function. Whereas the high frequency of ARF gene deletion in human cancers, accounting for some 40% of cancers overall, suggests that ARF would be a strong candidate for therapeutic application, the possible dependence of ARF activity on p53 and Rb function presents a potential limitation to its application, as these functions are often impaired in cancer. We show here that a replication-defective adenovirus, Ad1beta, encoding the exon 1beta region of ARF is most effective in tumor cells expressing endogenous wild-type p53. Nevertheless, Ad1beta suppresses tumor cell growth and viability in vitro and in vivo, inducing G1 or G2 cell cycle arrest and cell death even in tumor cells lacking both functional Rb and p53 pathways, and independently of induction of the p53 downstream targets, p21, bax, and mdm2. These results point to an activity of ARF in human tumor cells that is independent of Rb or p53, and suggest that therapeutic applications based on ARF would have a broad clinical application in cancer.