Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.