Purpose <p>Prostate-specific membrane antigen (PSMA) is a potential target for radioligand therapy (RLT) in neuro-oncology. This study investigates the direct relation between [⁶⁸Ga]Ga-PSMA-11 uptake on PET and PSMA expression in the tumour micro-environment of high-grade glioma (HGG) and brain metastasis (BM).</p> Methods <p>Twelve patients with HGG (glioblastoma <i>n</i> = 6, oligodendroglioma <i>n</i> = 1), or BM (lung- <i>n</i> = 4, breast cancer <i>n</i> = 1), underwent PET-MRI after intravenous [⁶⁸Ga]Ga-PSMA-11 injection (1.5&#xa0;MBq/kg), followed by image-guided biopsy sampling during (re-)resection surgery. Multiple samples (median <i>n</i> = 3/patient, <i>n</i> = 23 HGG/<i>n</i> = 20 BM) from locations of low and high [⁶⁸Ga]Ga-PSMA-11 uptake were analysed for PSMA expression in vasculature and non-vascular structures using morphology and immunohistochemistry.</p> Results <p>All patients showed [⁶⁸Ga]Ga-PSMA-11 uptake in tumour ( SUVmax median, range: 10.5, 4.7–19.8). Strong PSMA expression was found in tumour microvasculature (14/23, 61% in HGG, 13/20, 65% in BM). Tumour cell PSMA expression was found in a subset of HGG (10/23; of which strong in 8/10) and BM (3/20; none of which showed strong expression). Strong PSMA expression was also found on non-malignant glial cells in tumour. PSMA expression in healthy brain control samples was negligible. In HGG, a significant correlation existed between [⁶⁸Ga]Ga-PSMA-11 uptake and PSMA expression in tumour microvasculature (r = 0.487, <i>P</i> &lt; 0.01), but not tumour cells.</p> Conclusion <p>PSMA expression in brain tumours is predominately vascular, which likely explains why microvascular (rather than tumour cell) PSMA expression correlates with [⁶⁸Ga]Ga-PSMA-11 uptake in HGG. This neovascular expression is crucial information for future PSMA-based RLT studies, as alpha-emitters may not sufficiently target tumour DNA.</p> <p>NCT05798273; date of registration: 1/9/2020.</p>