Background: Epidemiologic studies have demonstrated that individuals who eat more fruits and vegetables and/or have high levels of serum β-carotene have a lower risk of cancer, especially lung cancer. However, recent human intervention studies using β-carotene supplements have shown an increase in the risk of lung cancer among smokers and asbestos workers. In this study, we used an animal model system to evaluate the hazard associated with a combination of high-dose β-carotene supplementation and tobacco smoking. Methods: Ferrets were given a β-carotene supplement, exposed to cigarette smoke, or both for 6 months. Cell proliferation and squamous metaplasia in lung tissue were assessed by examination of proliferatingcell nuclear antigen expression and histopathologic examination, respectively. β-Carotene and retinoid concentrations in lung tissue and plasma samples were analyzed by highperformance liquid chromatography. Expression of genes for retinoic acid receptors (RARs) and activator protein-1 (encoded by the c-Jun and c-Fos genes) in lung tissue specimens was examined by western blotting. Results: A strong proliferative response in lung tissue and squamous metaplasia was observed in all β-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared with control groups, all three treatment groups had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited 18%-73% reductions in RARβ gene expression; however, RARα and RARγ gene expression was not reduced. Ferrets given a β-carotene supplement and exposed to tobacco smoke had threefold to fourfold elevated expression of the c-Jun and cFos genes. Conclusions: Diminished retinoid signaling, resulting from the suppression of RARβ gene expression and overexpression of activator protein-1, could be a mechanism to enhance lung tumorigenesis after highdose β-carotene supplementation and exposure to tobacco smoke.