AbstractNeoxanthin, a major carotenoid pigment of spinach, is found in the chloroplast membrane and has an unknown function in plants. Neoxanthin inhibited the production of superoxide onions in an artificial xanthine and xanthine oxidase system and depressed DNA synthesis in methylcholanthrene (MCA)‐initiated C3H10T1/2 fibroblasts. In two‐stage carcinogenesis experiments, neoxanthin at 0.2μg/0.2 ml inhibited the formation of tumors that were induced sequentially by 7,12‐dimethylbenz[a]anthracene (DMBA) and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in the buccal pouch of Syrian Golden hamsters. To assess the ongoing process of carcinogenesis, the activity of ornithine decarboxylase (ODC), required for cell proliferation, was analyzed. Neoxanthin inhibited the activity of ODC when animals were treated with neoxanthin one hour before the application of TPA in two‐stage carcinogenesis. However, neoxanthin did not inhibit ODC activity when animals were treated with neoxanthin one hour before the application of DMBA in two‐stage carcinogenesis, and there was no subsequent tumor formation. In a short‐term anti‐initiation experiment, neoxanthin inhibited the covalent binding of isotope‐labeled DMBA to DNA by 53%. These results indicate that neoxanthin inhibits the initiation stage and the promotion stage in two‐stage carcinogenesis. This suggests that neoxanthin may act as a potential chemo‐preventive agent.