We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol.