NAD(P)H:quinone oxidoreductase 1 (NQO1) is implicated in both chemoprevention and bioactivation of DNA-damaging antitumor agents. NQO1 is mainly cytosolic, but distribution in other cellular compartments, particularly in tumor cells, is poorly defined. Nuclear NQO1 in HT29 human colon carcinoma and H661 human non-small cell lung cancer cells was observed using both confocal microscopy and immunoelectron microscopy. NQO1 was not detected in mitochondria, golgi, or endoplasmic reticulum. In addition, purified intact nuclei from HT29 cells contained immunoreactive NQO1, which was catalytically active as determined by conventional activity assay. In summary, we have confirmed the presence of nuclear NQO1, which has implications for chemoprotection and bioactivation of DNA-damaging antitumor agents.