We studied the influence of beta-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low beta-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans. beta-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by beta-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARbeta isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, beta-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.