The folate receptor (FR) has been established as a promising target for imaging and therapy of cancer (FR-α), inflammation, and autoimmune diseases (FR-β). Several folate based PET radiotracers have been reported in the literature, but an 18 F-labeled folate-PET imaging agent with optimal properties for clinical translation is still lacking. In the present study, we report the design and preclinical evaluation of folate-PEG 12 -NOTA-Al 18 F ( 1 ), a new folate-PET agent with improved potential for clinical applications. Radiochemical synthesis of 1 was achieved via a one-pot labeling process by heating folate-PEG 12 -NOTA in the presence of in situ prepared Al 18 F for 15 min at 105 °C, followed by HPLC purification. Specific binding of 1 to FR was evaluated on homogenates of KB (FR-positive) and A549 (FR-deficient) tumor xenografts in the presence and absence of excess folate. In vivo tumor imaging with folate-PEG 12 -NOTA-Al 18 F was compared to imaging with 99m Tc-EC20 using nu/nu mice bearing either KB or A549 tumor xenografts. Specific accumulation of 1 in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folate. Radiosynthesis of 1 was accomplished within ∼35 min, affording pure radiotracer 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ∼100% radiochemical purity after HPLC purification and a specific activity of 35.8 ± 15.3 GBq/mmol. Further in vitro and in vivo examination of 1 demonstrated highly specific FR-mediated uptake in FR+ tumor, with K d of ∼0.4 nM (KB), and reduced accumulation in liver. Given its facile preparation and improved properties, the new radiotracer, folate-PEG 12 -NOTA-Al 18 F ( 1 ), constitutes a promising tool for identification and classification of patients with FR overexpressing cancers.