| Summary: | Amino acid positron emission tomography (PET) has been employed in the management of brain metastases. Yet, histopathological correlates of PET findings remain poorly understood. We investigated the relationship of O-(2-[<sup>18</sup>F]Fluoroethyl)-L-tyrosine ([<sup>18</sup>F]FET) PET, magnetic resonance imaging (MRI), and histology in brain metastases. Fifteen patients undergoing brain metastasis resection were included prospectively. Using intraoperative navigation, 39 targeted biopsies were obtained from parts of the metastases that were either PET-positive or negative and MRI-positive or negative. Tumor and necrosis content, proliferation index, lymphocyte infiltration, and vascularization were determined histopathologically. [<sup>18</sup>F]FET PET had higher specificity than MRI (66% vs. 56%) and increased sensitivity for tumor from 73% to 93% when combined with MRI. Tumor content per sample increased with PET uptake (r<sub>s</sub> = 0.3, <i>p</i> = 0.045), whereas necrosis content decreased (r<sub>s</sub> = −0.4, <i>p</i> = 0.014). PET-positive samples had more tumor (median: 75%; interquartile range: 10–97%; <i>p</i> = 0.016) than PET-negative samples. The other investigated histological properties were not correlated with [<sup>18</sup>F]FET PET intensity. Tumors were heterogeneous at the levels of imaging and histology. [<sup>18</sup>F]FET PET can be a valuable tool in the management of brain metastases. In biopsies, one should aim for PET hotspots to increase the chance for retrieval of samples with high tumor cell concentrations. Multiple biopsies should be performed to account for intra-tumor heterogeneity. PET could be useful for differentiating treatment-related changes (e.g., radiation necrosis) from tumor recurrence.
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