Suppression of tumorigenesis is considerably more stringent in the human than in the much shorter-lived mouse species, and the reasons for this difference are poorly understood. We investigated functional differences in the control of the ARF (Alternative Reading Frame) protein that acts upstream of p53 and is encoded along with p16INK4a at a major tumor suppressor locus in both the human and mouse genomes. The mouse and human ARF proteins are substantially divergent at their C-termini. We have shown that the mouse ARF protein (p19ARF) interacts with Pex19p in the cell cytoplasm leading to its nuclear exclusion and repression of its p53 activation function. The human ARF protein (p14ARF) is substantially smaller than its mouse counterpart and is not subject to this functional inactivation by Pex19p. In an identical cellular background, ribozymes directed against Pex19p enhanced p19ARF-, but not p14ARF-activated p53 function. This is the first demonstration of a functional difference between the mouse and human ARF proteins. In view of the major role of ARF in tumor suppression, this distinction may contribute to the different levels of tumor-proneness of these species.