Lycopene (LYC) is the major tomato carotenoid and is the focus of substantial research. Phytoene (PE), a minor tomato carotenoid, is found in human blood and tissues in similar concentrations to LYC. To determine which metabolic differences underlie this phenomenon, Mongolian gerbils (Meriones unguiculatus, n = 56) were fed control or tomato powder (TP)-containing diets (to establish steady-state serum and tissue carotenoid concentrations similar to tomato-fed humans) for 26 d. The TP-fed gerbils were then provided either a single, oral, cottonseed oil (CO) vehicle dose and tissues were collected at 6 h or they were provided unlabeled PE or LYC in CO and tissues were evaluated at 6, 12, or 24 h. In vehicle-dosed, TP-fed gerbils, LYC was the major carotenoid (≥ 55% carotenoids) in liver, spleen, testes, and the prostate-seminal vesicle complex, whereas PE was the major serum and adipose carotenoid (≥ 37% total carotenoid) and phytofluene was the major carotenoid (≥ 38%) in adrenals and lungs. PE dosing increased hepatic, splenic, and serum PE concentrations compared with vehicle dosing (P < 0.05) from 6 to 24 h, whereas LYC dosing increased only serum LYC at 6 and 12 h (P < 0.05) compared with vehicle dosing. This suggested PE was more bioavailable and cleared more slowly than LYC. To precisely track absorptive and distributive differences, (14)C-PE or (14)C-LYC (n = 2/group) was provided to TP-fed gerbils. Bioavailability assessed by carcass (14)C-content was 23% for PE and 8% for LYC. Nearly every extra-hepatic tissue accumulated greater dose radioactivity after (14)C-PE than (14)C-LYC dosing. Thus, LYC and PE, which structurally differ only by saturation, pharmacokinetically differ in bioavailability, tissue deposition, and clearance.