Tomato product consumption is inversely related to prostate cancer incidence, and lycopene (LYC) has been implicated in reduced prostate cancer risk. The contribution of other tomato carotenoids, phytoene (PE) and phytofluene (PF), toward prostate cancer risk has not been adequately studied. The relative uptake and tissue distribution of tomato carotenoids are not known. We hypothesize that PE and PF are bioavailable from a tomato powder diet or from a purified source and accumulate in androgen-sensitive tissues. In this study, 4-week-old male Fisher 344 rats (Harlan, Indianapolis, Ind) were prefed an AIN-93G powder diet composed of 10% tomato powder containing PE, PF, and LYC (0.015, 0.012, and 0.011 g/kg diet, respectively). After 30-day tomato powder feeding, hepatic PF concentrations (168 ± 20 nmol/g) were higher than PE or LYC (104 ± 13 and 104 ± 13 nmol/g, respectively). In contrast, LYC, followed by PF, had the highest accumulation of the measured carotenoids in the prostate lobes and seminal vesicles. When tomato powder–fed rats received a single oral dose of either approximately 2.7 mg PE or approximately 2.7 mg PF, an increase in the dosed carotenoid concentration was observed in all measured tissues, except in the adrenal. The percentages of increases of PF were greater than that of PE in liver, serum, and adipose (37%, 287%, and 49% vs 16%, 179%, and 23%, respectively). Results indicate that the relative tomato-carotenoid biodistribution differs in liver and androgen-sensitive tissues, suggesting that minor changes in the number of sequential double bonds in carotenoid structures alter absorption and/or metabolism of tomato carotenoids.