Fluorescence emission, polarization and subcellular localization of methylene blue (MB) were studied in four cancerous and two normal human brain cell lines. Fluorescence emission and polarization images were acquired and analyzed. The co-localization of MB with mitochondria, lysosomes and nuclei of the cells was evaluated. Glioblastoma cells exhibited significantly higher MB fluorescence polarization compared to normal astrocytes. Preferential accumulation of MB in mitochondria of glioblastoma cells may explain higher fluorescence polarization values in cancer cells as compared to normal. These findings may lead to the development of a quantitative method for the detection of brain cancer in single cells.