DNA polymerase κ (POLκ) is a low fidelity translesional DNA polymerase implicated in spontaneous and DNA damage-induced mutagenesis. We have previously shown that POLκ was frequently overexpressed in human lung cancer tissues as compared with their matched nontumorous tissue counterpart. In the present study, we found a close correlation between elevated POLκ expression and p53 inactivation in lung cancer tissues. To investigate whether POLK expression might be regulated by p53, we have determined the transcriptional initiation site of POLK gene and examined its promoter activity in A549, H358-129, and PC-3 human lung cancer cell lines. Wild-type p53, but not a mutant p53 (R273H) devoid of the DNA-binding activity, strongly inhibited POLK promoter activity in these cells. In addition, POLK promoter exhibited a significantly higher activity in p53-/- murine embryo fibroblasts (MEF) than in p53+√- and p53+√+ MEF. These results link p53 status with POLκ expression and suggest that loss of p53 function may in part contribute to the observed POLκ upregulation in human lung cancers.