Consumption of tomatoes and tomato carotenoids is associated with a reduced risk of prostate cancer. Prostate tissue accumulates tomato carotenoids, including lycopene, beta-carotene, and phytoene. Phytoene accumulation is relatively greater in the prostate than that of lycopene, but the metabolic determinants of tissue carotenoid profiles are poorly understood. The purpose of this study was to determine if differences in stability, cellular uptake, and clearance of phytoene versus lycopene or beta-carotene by prostate and intestinal cells may explain differences in observed tissue carotenoid profiles. Gene and protein expression for carotenoid metabolism in prostate cell lines were analyzed by qRT-PCR and Western blot, respectively. Uptake, efflux, and clearance of phytoene, lycopene, or beta-carotene by prostate cell (LNCaP, RWPE-1, and PC-3) and absorptive enterocyte (Caco-2) cultures were compared. The effect of scavenger receptor class B member 1 (SCARB1) inhibition on carotenoid uptake by LNCaP, RWPE-1, and Caco-2 cells was tested. SCARB1 was expressed across prostate cell lines. Lycopene, phytoene, and beta-carotene uptake were similar in LNCaP and PC-3 cells, while RWPE-1 cells absorbed a smaller portion of the phytoene dose than lycopene or beta-carotene doses. The clearance rates of carotenoids from LNCaP cells did not differ. Intestinal cell uptake of phytoene was greatest, followed by beta-carotene and lycopene. SR-BI inhibitor treatment did not significantly reduce the uptake or efflux of carotenoids by LNCaP or Caco-2 cells at the dose level provided. Overall, this study suggests that greater bioavailability at the point of the intestine and greater stability of phytoene are determinants of the relative enrichment of phytoene in prostate tissue.