Microdialysis enables measurement of the chemistry of the cerebral extracellular fluid. This study's objective was to utilise microdialysis to monitor levels of glucose, lactate, pyruvate, glutamate and glycerol in patients following surgery for intrinsic brain tumours, and to assess the concentration of growth factors, cytokines and other proteins involved in the pathogenesis of high-grade gliomas in vivo. Eight patients with suspected high-grade gliomas were studied. Seven of these underwent resection with one microdialysis catheter placed at the tumour resection margin and, in six of these seven cases, a second microdialysis catheter in macroscopically normal peritumour tissue. The remaining glioma patient had an image-guided biopsy with a single catheter inserted stereotactically at the tumour margin. Histology demonstrated WHO IV glioblastoma in five cases, WHO III anaplastic astrocytoma in two cases, and one cerebral lymphoma. In the high-grade gliomas (WHO IV and III), tumour margin microdialysates consistently showed significantly lower glucose, higher lactate/pyruvate (L/P) ratio, higher glutamate and higher glycerol, relative to peritumour microdialysates (P < 0.05). These results indicate that malignant glioma margin tissue is metabolically extremely active. There was great variability in the microdialysate concentrations of growth factors (TGFalpha, EGF, VEGF), cytokines (IL-1alpha, IL-1beta, IL-1ra, IL-6, IL-8), matrix metalloproteinases (MMP-2, MMP-9) and their endogenous inhibitors (TIMP-1, TIMP-2). Notably, microdialysates from the glioma resection margin demonstrated significantly higher IL-8 concentration and higher MMP-2/TIMP-1 ratio when compared to peritumour microdialysates (P < 0.05), suggesting an environment favouring invasion and angiogenesis at the tumour margin. Microdialysis is a promising technique to study in vivo glioma metabolism, and may assist in the development of new therapies.