OBJECTIVE Glioma heterogeneity and the limitations of conventional structural MRI for identifying aggressive tumor components can limit the reliability of stereotactic biopsy and, hence, tumor characterization, which is a hurdle for developing and selecting effective treatment strategies. In vivo MR spectroscopy (MRS) and PET enable noninvasive imaging of cellular metabolism relevant to proliferation and can detect regions of more highly active tumor. Here, the authors integrated presurgical PET and MRS with intraoperative neuronavigation to guide surgical biopsy and tumor sampling of brain gliomas with the aim of improving intraoperative tumor-tissue characterization and imaging biomarker validation. METHODS A novel intraoperative neuronavigation tool was developed as part of a study that aimed to sample high-choline tumor components identified by multivoxel MRS and (18)F-methylcholine PET-CT. Spatially coregistered PET and MRS data were integrated into structural data sets and loaded onto an intraoperative neuronavigation system. High and low choline uptake/metabolite regions were represented as color-coded hollow spheres for targeted stereotactic biopsy and tumor sampling. RESULTS The neurosurgeons found the 3D spherical targets readily identifiable on the interactive neuronavigation system. In one case, areas of high mitotic activity were identified on the basis of high (18)F-methylcholine uptake and elevated choline ratios found with MRS in an otherwise low-grade tumor, which revealed the possible use of this technique for tumor characterization. CONCLUSIONS These PET and MRI data can be combined and represented usefully for the surgeon in neuronavigation systems. This method enables neurosurgeons to sample tumor regions based on physiological and molecular imaging markers. The technique was applied for characterizing choline metabolism using MRS and (18)F PET; however, this approach provides proof of principle for using different radionuclide tracers and other MRI methods, such as MR perfusion and diffusion.