Death receptor 4 (DR4) is one of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and triggers apoptosis on ligation with TRAIL or overexpression. Our previous study demonstrated that DR4 expression could be regulated in a p53-dependent fashion. In the present study, we have demonstrated that DR4 is a p53 target gene and is regulated by p53 through a functional intronic p53 binding site (p53BS) based on the following lines of evidence: (a) the p53BS in the DR4 gene is almost identical to the one found in the first intron of the DR5 gene in terms of their locations and sequences; (b) DR4 p53BS bound to p53 protein in intact cells upon p53 activation as demonstrated by a chromatin immunoprecipitation assay; (c) a luciferase reporter vector carrying the DR4 p53BS upstream of an SV40 promoter exhibited enhanced luciferase activity when transiently cotransfected with a wild-type p53 expression vector in p53-null cell lines or stimulated with DNA-damaging agents in a cell line having wild-type p53; and (d) when the DR4 p53BS, together with its own corresponding promoter region in the same orientation as it sits in its natural genomic locus, was cloned into a basic luciferase vector without a promoter element, its transcriptional activity was strikingly increased by cotransfection of a wild-type p53 expression vector or treatment with DNA-damaging agents. However, wild-type p53 or DNA-damaging agents completely lost their activity to increase transcriptional activity of a reporter construct with deleted DR4 p53BS. Thus, we conclude that p53 directly regulates the expression of the DR4 gene via the novel intronic p53BS.