The radiosensitivity of the human glioma cell line U-343MGa, while growing as spheroids and as conventional monolayers, was studied. The spheroids were first irradiated with 60Co photons, and the radiosensitivity was then analyzed in different cell layers with varying proliferative activity. The different cell layers were isolated by automated sequential trypsinization. A relatively high radioresistance was found in all analysed cell layers, and the inner, mainly quiescent cells, were as resistant as the outer proliferating cells. It was, in parallel experiments on monolayers, shown that serum starved quiescent cells were as resistant as proliferating cells, and that direct cellular cloning did not give lower cell survival values than delayed cloning, as has been shown for other cell types. Thus, the studied glioma cells were equally radioresistant under all tested proliferative conditions when conventional low-LET radiation was applied. The spheroids were then irradiated with cyclotron accelerated helium ions with an intermediate ionization density, LET approximately equal to 55 keV/microns, and this gave a clear decrease in survival in all cell layers.