The ARF (p19(ARF) for the mouse ARF consisting of 169 amino acids and p14(ARF) for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19(ARF). We constructed various deletion mutants of p19(ARF) with a cre/loxP-regulated adenoviral vector to determine the regions of P19(ARF) which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19(ARF) whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19(ARF) 1-129, a P19(ARF) deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14(ARF) did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution. (C) 2003 Elsevier Science (USA). All rights reserved.