Cells obtained from a human glioblastoma (G5) were characterized and used to develop an assay measuring their radiosensitivity in vitro. Surviving fractions were estimated 12 days after irradiation by image analysis of the total surface occupied by the cells. This report evaluates 4 experimental factors which may influence the radiosensitivity in vitro of G5 cells: passage number, delay between plating and irradiation, cell density and clonal heterogeneity. The radiosensitivity of the G5 cell line was found to be passage-independent at least between passages 12 and 75. Experimental conditions influence the radiosensitivity as surviving fraction at 2 Gy (SF2) range from 90% (5,000 cells/well, irradiation 72 h after seeding) to 49% (2,500 cells per well, irradiation 24 h after seeding). The heterogeneity of the radiosensitivity is large at the clonal level as SF2 of six clones isolated from the G5 line were 45%, 50%, 72%, 74%, 79% and 84%. Finally, when G5 cells were irradiated at low cell density and at the beginning of the growth phase, the radiosensitivity measured with this assay is comparable to that obtained with a standard colony assay. We propose that this assay may be useful to determine the intrinsic radiosensitivity of cells obtained from human tumours.