During 5-aminolevulinic acid (ALA)-guided glioblastoma multiforme (GBM) surgery, we encountered fluorescence in ventricular walls that lacked enhancement on magnetic resonance (MR) images and were free of macroscopic invasion of tumor cells. However, the meaning of ventricular wall fluorescence during 5-ALA-guided surgery is still unknown. The aim of this study was to investigate the relationship between intraoperative 5-ALA fluorescence and histopathological findings of ventricular walls free of enhancement on MR images. Nineteen patients with newly diagnosed GBM located near the lateral ventricle underwent 5‑ALA fluorescence‑guided surgery. During the surgery, the ventricle wall was opened and investigated with the aid of a surgical microscope equipped with optical filters to examine 5‑ALA fluorescence of the ventricular wall. Twenty‑five ventricular wall tissues that were apparently free of tumor involvement by MR imaging and macroscopic observation were obtained during surgery. Among the 19 cases with brightly fluorescing tumor masses, 11 patients (57.9%) exhibited 5‑ALA‑induced fluorescence in the ventricular wall. Of the 25 ventricular wall samples, 11 exhibited 5‑ALA‑induced fluorescence; upon pathologic examination, tumors were present in 5 samples (45.5%), but the remaining 6 (54.5%) were free of tumor cells. A pathologic examination revealed no tumor cells in the 14 samples that lacked 5‑ALA‑induced fluorescence. Our data suggest the possibility that glioma cells exhibiting 5‑ALA fluorescence are present in the ventricle wall, despite no signs of tumor involvement in MR images. Further investigation of non‑tumor cells from tissues with 5‑ALA fluorescence is needed to understand the nature of this unexpected ventricular wall fluorescence.