We show that the expression of the human cytokeratin 8 (CK8) gene is regulated by wild-type p53. DNA sequence data indicate that the 5' untranslated region of the CK8 gene contains a putative p53-like binding site. In this study we focused on the effect of the p53 protein on the regulation and expression of the CK8 gene. Cotransfection of the H358 p53-negative human lung cancer cell line with a CK8 promoter CAT expression vector and a plasmid expressing the wildtype p53 indicated that p53 induces CK8 expression. A transient assay in which a p53-negative cell line was cotransfected with a CK8 promoter CAT expression vector and a plasmid expressing wildtype or mutant p53 indicated that only wildtype p53 induces the CK8 promoter. Deletion of the putative p53-binding site from the CK8 promoter or introduction of mutations in the p53-binding sequences abolished the wild-type p53-mediated transactivation of CK8. A gel-retardation assay was used to measure DNA binding by the wild-type p53 protein. A 24-bp oligonucleotide corresponding to the putative p53 binding site was used for this assay. The wild-type p53 protein bound weakly to this DNA sequence but much more strongly when three tandem repeat of the binding sequences was used. These studies suggest that the CK8 gene is a downstream target whose expression is regulated by wild-type p53.