The in vivo high-energy phosphorus metabolic profile and pH of an experimental intracerebral C6 glioma in rats was examined using surface coil 31P NMR spectroscopy. Initially, phosphorus-containing metabolites of the glioma were characterized by in vivo 31P surface coil spectroscopy of subcutaneously implanted tumors and by high-resolution NMR studies of perchloric acid (PCA) extracts of both freeze-clamped subcutaneous tumor tissue and cultured cells. These studies demonstrated that the C6 glioma has reduced levels of phosphocreatine (PCr) compared to the levels found in normal rat brain. Thus, reduced spectral PCr levels were useful as a metabolic indicator for monitoring the spatial selectivity of tumor metabolism distinct from that of adjacent normal brain tissue. To maximize 31P NMR signals from intracerebral tumors, tumor cells were stereotaxically placed superficially in the brain. Proton magnetic resonance imaging (1H MRI) was used to determine the size and location of the resultant brain tumors in order to preselect rats with large superficial tumors for spectroscopic study. 31P NMR spectra of the glioma tumors revealed a consistent reduction in the PCr/ATP ratio, an increase in the Pi/ATP ratio, and a slightly increased tissue pH. No correlation was found between levels of Pi/ATP and tumor pH in subcutaneous or intracerebral gliomas and the amount of necrosis as determined histologically. This study demonstrates that phosphorus metabolites of an experimental brain tumor in the rat can be monitored in vivo with minimal contributions from adjacent normal brain tissue metabolites using surface coil 31P NMR spectroscopy.