Abstract. The poor prognosis of patients suffering from malignant glioma requires further efforts. Photodynamic therapy (PDT) might be a therapeutic option to increase surgical radicality. Hypericin (HY) exhibit high phototoxicity to malignant cells and accumulates to a higher extent in glioblastoma cells as compared to neurons. Therefore, the impact of various experimental parameters on cytotoxicity, intracellular accumulation and phototoxicity of HY was quantitatively assessed in the three human glioblastoma cell lines U373 MG, LN229 and T98G. Additionally, intracellular location of HY was studied with fluorescence microscopic techniques. For all three cell lines, no cytotoxicity was found for incubation concentrations up to 5 μM. For short-time incubation (2 h), maximum HY fluorescence was achieved at an incubation concentration of about 5 μM. However, uptake kinetics of HY was dependent on its incubation concentration. Moreover, increase in HY fluorescence was negligible at 4˚C, which strongly indicates that the compound is taken up by an energy-dependent process. HY exhibited high phototoxicity (at 595 nm) in all three cell lines with ID 50 -values ranging from 0.15 J/cm 2 to 0.22 J/cm 2 , but sensitivity decreased in the order U373 MG > LN229 > T98G. However, assessment of phototoxicity at different wavelengths revealed that highest cell inactivation was achieved at 600 nm. Fluorescence microscopy showed that HY fluorescence arose predominantly from the perinuclear region and the nuclear membrane. Fluorescence pattern of HY was significantly different from those observed for organelle markers staining lysosomes or mitochondria. Location of HY in the plasma membrane was proven by total internal reflection fluorescence microscopy. Thus, the present study demonstrates that glioblastoma cells can be effectively inactivated by HY-PDT after short-time incubation and exposure to low light doses. These results obtained in cell culture are encouraging and justify further evaluation HY-PDT for the treatment of malignant glioma in animal experiments.