The apoptotic function of N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was investigated in cultured mammalian cells. TPCK induced apoptosis in Jurkat, which was not accompanied with DNA fragmentation. A treatment of TPCK resulted in the activation of JNK and p38 kinase pathway in a variety of cell lines, followed by a phosphorylation of its downstreatm targets. TPCK-induced apoptosis was shown to be p53-dependent in HCT116 p53-/- cells. Among caspase family tested, caspase-3 and caspase-7 were processed upon TPCK treatment in HCT116 p53+/+ cells, while no cleavage of caspases was observed in its p53 deficient counterpart. TPCK promoted dephosphorylation of p53 on serine residues at 6, 9, 46, 376 and 378 in parallel with the activation of p53 transcriptional activity in a promoter specific manner. TPCK also induced p53 condensation in nucleus which was due to an association with PML which is a key recruiter of nuclear structure called NB or POD. Cells expressing p53 mutant in which serine residues at 6, 9, 46, 376 and 378 were replaced by aspartic acid were resistant to TPCK-induced apoptosis. Transfection of this mutant p53 also interfered with transcriptional activation of p53, caspase cleavage, and colocalization of p53 with PML induced by TPCK, suggesting the requirement of dephosphorylation of p53 during these processes.