Phenethylisothiocyanate (PEITC), a potential cancer chemopreventive agent, induces colon cancer cell death, but the mechanism is not entirely clear. Therefore, the aim of this study was to further clarify the molecular effects of PEITC in causing death of human colon adenocarcinoma cells. When incubated with PEITC, HCT-116 colonocytes showed morphological features characteristic of apoptosis, such as irregular cell shape, translocation of plasma membrane phosphatidylserine, and also chromatin condensation and fragmentation. These changes occurred after single-strand breaks in DNA were detected, suggesting that PEITC induced irreparable DNA damage, which in turn triggered the process of apoptosis. DNA macroarray analysis of a selected small cluster of apoptosis-related genes revealed noticeably higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. This led to investigation of other GADD gene members possibly affected by PEITC. Whereas GADD34 mRNA expression increased just slightly, there was an appreciable elevation of the mRNA for GADD153, which is recognized as a pro-apoptotic gene. The effect of PEITC on GADD153 was attenuated by either actinomycin D or N-acetylcysteine, suggesting that PEITC-induced upregulation of GADD153 mRNA expression was partly at the level of transcriptional activation involving reactive oxygen species. Additionally, PEITC-induced upregulation of GADD153 mRNA expression did not appear to require p53, based on the observation that PEITC also increased GADD153 mRNA expression in HCT-15 colonocytes, which are known to express mutant p53. These findings suggest that PEITC creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis. © 2003 Wiley-Liss, Inc.