Prostate cancer is characterized by a low rate of glycolysis and glucose uptake. We hypothesize that fatty acid is dominant over glucose in uptake by prostate cells. One benign (RWPE1) and two malignant (LNCaP and PC3) prostate cell lines were assayed for their in vitro uptake of radiolabeled glucose analogs ³H-fluoro-2-deoxyglucose and ¹⁸F-fluoro-2-deoxyglucose, and the long-chain fatty acid ³H-palmitic acid. Fractional uptake was standardized to viable cell numbers. Uptake of palmitate in all 3 prostate cell lines was significantly higher than that of glucose at all incubation times (p<0.01). But in malignant cell lines, neither glucose nor palmitate uptake was quantitatively higher than that in the benign cell line. The uptake of fatty acid by prostate cells is a dynamic, active process mediated by the membrane receptors. Prostate cells are characterized by a dominant uptake of fatty acid over glucose, suggesting that future development of new diagnostic and therapeutic approaches in prostatic cancer should focus on fatty acid substrate. Fatty acid imaging may be useful in detection of recurrence and metastasis, but not in differentiating malignant from benign prostate tissue.