Penclomedine, a synthetic pyridine derivative, has documented antitumor activity and is being investigated in clinical trials. Its mechanism of action is unknown although it may be metabolized to a free radical, DNA-reactive species. We previously reported that telomerase positive colorectal carcinoma (RKO) cells with abrogated p53 function were more sensitive to penclomedine than were telomerase positive cells with wild-type p53. The present study demonstrates that significant differences in DNA fragmentation in response to penclomedine were observed in RKO cells lacking functional p53 compared with RKO cells with normal p53 function. No differences in DNA fragmentation in response to ionizing radiation were seen in RKO cells with normal or abrogated p53 function. RKO cells with functional p53 respond to penclomedine treatment with a dose-dependent increase in p53 protein levels. However, RKO cells with abrogated p53 function did not show any such change in p53 protein levels. Further, p53-independent increase of p21 was observed, although the significance of this response remains uncertain. These studies suggest that penclomedine may have a therapeutic advantage in killing cells that have abrogated p53 function.