Thirty consecutive surgical patients with glioblastoma, were operated upon using fluorescence induced by 5-aminolevulinic acid as guidance. The fluorescent quality of the tissue was used to take biopsies from the tumor center, from the invasive area around it and from adjacent normal-looking tissue. These samples were analyzed with HE, Ki-67 and nestin. Nestin expression in tissue surrounding glioblastoma cases was compared to tissue surrounding vascular lesions, metastasis and hippocampal sclerosis. The rate of gross total resection assessed by volumetric MRI was 83%. Using HE examination as the gold standard, fluorescence identified solid tumor with 100% positive predictive value, invasive areas with 97%, and normal tissue with 67% negative predictive value. Ki67 stained some cells in 69% of the non-fluorescent samples around the tumor. There was always strong nestin expression around the tumor but it was similar to control cases in non-glioma lesions with subacute expansion. 5-aminolevulinic acid fluorescence guidance is very reliable and can help to study the tumor-brain interface. Nestin expression is strong and constant in the tissue around the tumor, but is mostly an acute glial reaction, not specific of the neoplasm. Nestin staining is not recommended as a tumor stem cell marker.