It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal (31)RRPR(34) did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.