Purpose: The preclinical evaluation of 3-l- and 3-d-[18F]FPhe in comparison to [18F]FET, an established tracer for tumor imaging. Methods: In vitro studies were conducted with MCF-7, PC-3, and U87 MG human tumor cell lines. In vivo µPET studies were conducted in healthy rats with/without the inhibition of peripheral aromatic l-amino acid decarboxylase by benserazide pretreatment (n = 3 each), in mice bearing subcutaneous MCF-7 or PC-3 tumor xenografts (n = 10), and in rats bearing orthotopic U87 MG tumor xenografts (n = 14). Tracer accumulation was quantified by SUVmax, SUVmean and tumor-to-brain ratios (TBrR). Results: The uptake of 3-l-[18F]FPhe in MCF-7 and PC-3 cells was significantly higher relative to [18F]FET. The uptake of all three tracers was significantly reduced by the suppression of amino acid transport systems L or ASC. 3-l-[18F]FPhe but not 3-d-[18F]FPhe exhibited protein incorporation. In benserazide-treated healthy rats, brain uptake after 42–120 min was significantly higher for 3-d-[18F]FPhe vs. 3-l-[18F]FPhe. [18F]FET showed significantly higher uptake into subcutaneous MCF-7 tumors (52–60 min p.i.), while early uptake into orthotopic U87 MG tumors was significantly higher for 3-l-[18F]FPhe (SUVmax: 3-l-[18F]FPhe, 107.6 ± 11.3; 3-d-[18F]FPhe, 86.0 ± 4.3; [18F]FET, 90.2 ± 7.7). Increased tumoral expression of LAT1 and ASCT2 was confirmed immunohistologically. Conclusion: Both novel tracers enable accurate tumor delineation with an imaging quality comparable to [18F]FET.