The underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) exert a chemopreventive effect in the colon have not been elucidated. Retinoid X receptors (RXR) are a family of nuclear receptors implicated in cancer chemoprevention. Since docosahexaenoic acid (DHA), an n-3 PUFA enriched in fish oil, reduces colonocyte proliferation and enhances apoptosis relative to n-6 PUFA-treated cells, we determined whether DHA can serve as a specific ligand for RXRα activation relative to n-6 PUFA in colonocytes. In a mammalian one-hybrid assay, immortalized young adult mouse colonic (YAMC) cells were co-transfected with a yeast galactose upstream activating sequence (UAS) ₄ -tk-Luciferase (Luc) reporter plasmid, plus either GAL4 DNA-binding domain fused to RXRα, retinoic acid receptor α or GAL4 alone, followed by an n-3, n-6 or n-9 fatty acid incubation. Luc activity levels were dose-dependently elevated only in n-3 PUFA (DHA)-treated RXRα. Since RXR homodimers and RXR/peroxisome proliferator-activated receptor (PPAR) heterodimers bind consensus direct repeat (DR1) motifs, YAMC and NCM460 (a normal human colonic cell line), were respectively, co-transfected with RXRα and DR1-Luc, followed by different PUFA treatment. Luc activity levels were increased ( P < 0.05) only in DHA groups. The DHA-dependent induction of DR-1-Luc was reduced to basal levels upon RXRα antagonist-treatment, with no effect on PPARγ antagonist-treatment. A role for select RXR isoforms in colonocyte biology was also determined by examining nuclear receptor mRNA levels in rat colon following dietary lipid and carcinogen exposure over time. RXRα, RXRβ and RXRγ were detected in rat colonic mucosa, and the levels of RXRα and RXRγ were elevated in fish oil (n-3 PUFA) versus corn oil (n-6 PUFA) fed animals after 16 weeks. These data indicate that, RXRα, an obligatory component of various nuclear receptors, preferentially binds n-3 PUFA in colonocytes, and that the nuclear receptor targets for PUFA in the colon are modulated by dietary lipid exposure.