MRI is commonly used to determine the location and extent of cerebral gliomas. We investigated whether the diagnostic accuracy of MRI could be improved by the additional use of PET with the amino acid O-(2-[18F]fluoroethyl)-l-tyrosine (FET). In a prospective study, PET with FET and MRI was performed in 31 patients with suspected cerebral gliomas. PET and MRIs were co-registered and 52 neuronavigated tissue biopsies were taken from lesions with both abnormal MRI signal and increased FET uptake (match), as well as from areas with abnormal MR signal but normal FET uptake or vice versa (mismatch). Biopsy sites were labelled by intracerebral titanium pellets. The diagnostic performance for the identification of cellular tumour tissue was analysed for either MRI alone or MRI combined with FET PET using alternative free response receiver operating characteristic curves (ROCs). Histologically, 26 biopsy samples corresponded to cellular glioma tissue and 26 to peritumoral brain tissue. The diagnostic performance, as determined by the area under the ROC curve (Az), was Az = 0.80 for MRI alone and Az = 0.98 for the combined MRI and FET PET approach (P < 0.001). MRI yielded a sensitivity of 96% for the detection of tumour tissue but a specificity of only 53%, and combined use of MRI and FET PET yielded a sensitivity of 93% and a specificity of 94%. Combined use of MRI and FET PET in patients with cerebral gliomas significantly improves the identification of cellular glioma tissue and allows definite histological tumour diagnosis. Thus, our findings may have considerable impact on target selection for diagnostic biopsies as well as therapy planning.