Upregulation of prostate-specific membrane antigen (PSMA) in neovasculature has been described in glioblastoma multiforme (GBM), whereas vasculature in nonaffected brain shows hardly any expression of PSMA. It is unclear whether PSMA-targeting tracer uptake on PET is based on PSMA-specific binding to neovasculature or aspecific uptake in tumor. Here, we quantified uptake of various PSMA-targeting tracers in GBM and correlated this with PSMA expression in tumor biopsy samples from the same patients. Methods: Fourteen patients diagnosed with de novo (n 5 8) or recurrent (n 5 6) GBM underwent a preoperative PET scan after injection of 1.5 MBq/kg [ 68 Ga]Ga-PSMA-11 (n 5 7), 200 MBq of [ 18 F]DCFpyl (n 5 3), or 200 MBq of [ 18 F]PSMA-1007 (n 5 4). Uptake in tumor and tumor-to-background ratios, with contralateral nonaffected brain as background, were determined. In a subset of patients, PSMA expression levels from different regions in the tumor tissue samples (n 5 40), determined using immunohistochemistry (n 5 35) or RNA sequencing (n 5 13), were correlated with tracer uptake on PET. Results: Moderate to high (SUV max , 1.3-20.0) heterogeneous uptake was found in all tumors irrespective of the tracer type used. Uptake in nonaffected brain was low, resulting in high tumor-tobackground ratios (6.1-359.0) calculated by dividing SUV max of tumor by SUV max of background. Immunohistochemistry showed variable PSMA expression on endothelial cells of tumor microvasculature, as well as on dispersed individual cells (of unknown origin), and granular staining of the neuropil. No correlation was found between in vivo uptake and PSMA expression levels (for immunohistochemistry, r 5 20.173, P 5 0.320; for RNA, r 5 20.033, P 5 0.915). Conclusion: Our results indicate the potential use of various PSMA-targeting tracers in GBM. However, we found no correlation between PSMA expression levels on immunohistochemistry and uptake intensity on PET. Whether this may be explained by methodologic reasons, such as the inability to measure functionally active PSMA with immunohistochemistry, tracer pharmacokinetics, or the contribution of a disturbed blood-brain barrier to tracer retention, should still be investigated.