9-cis-Retinoic acid (9cRA) and 1α,25-dihydroxyvitamin D3 (1,25D) show promise as potential chemopreventive agents. We examined 9cRA and 1,25D, alone and in combination, for their potential to inhibit carcinogen (NNK)-induced lung carcinogenesis in A/J mice. A/J mice (n = 14/group) were treated with 9cRA (7.5, 15, or 30 mg/kg diet), 1,25D (2.5 or 5.0 μg/kg diet), or a combination of 9cRA (15 mg/kg diet) plus 1,25D (2.5 μg/kg diet) for 3 weeks before and 17 weeks after carcinogen injection. Lung tumor incidence, tumor multiplicity, plasma 1,25D levels and kidney expression of vitamin D 24-hydroxylase (CYP24) were determined. Compared to carcinogen-injected controls, mice receiving 9cRA supplementation had significantly lower tumor multiplicity at all doses (decreased 68–85%), with body weight loss at the higher doses of 9cRA. Mice receiving 1,25D supplementation had significantly lower tumor incidence (decreased 36 and 82%) and tumor multiplicity (decreased 85 and 98%), but experienced significant body weight loss, kidney calcium deposition, elevated kidney CYP24 expression and decreased fasting plasma 1,25D levels. Although, there was no apparent influence on chemopreventive efficacy, addition of 9cRA to 1,25D treatment effectively prevented the weight loss and kidney calcification associated with 1,25D treatment alone. These data demonstrate that 9cRA and 1,25D, alone or combined, can inhibit lung tumor promotion in the A/J mouse model. Combining 1,25D with 9cRA has the potential to mitigate the toxicity of 1,25D, while preserving the significant effect of 1,25D treatment against lung carcinogenesis. The underlying mechanism behind this effect does not appear to be related to retinoid modulation of vitamin D catabolism. © 2006 Wiley-Liss, Inc.