Cells of the established HT29 human colon adenocarcinoma line were grown as spheroids using continuous machine sstirring of cells in flasks coated with agarose. The cell response for different layers of the spheroids and a non-confluent monolayer culture to 220 kV x-rays was studied. A successful protocol was developed for the separation of HT29 spheroids into four concentric layers with trypsinization by manual stirring. The cell response for different layers of the spheroids and a non-confluent monolayer culture to 220 kV x-rays was studied. From the extracted cells of different layers cell survival was measured by maintenance of colony-forming ability for cells seeded either immediately after or 18h after irradiation. The LQ model was fitted to the survival data. Each of the layers had an oxygen level associated to it, as determined by a previously published oxygen profile for spheroids of cells from this cell line. In addition, cell-cycle arrest and in situ repair effects 18 hours after irradiation were studied for the same spheroid layers, with survival curves and DNA histograms. Staining of fixated and sliced spheroids with the H\&E immunohistochemical agent revealed two discrete regions: an outer region of viable cells close to the rim and a core of necrotic cells. The thicknesses of the trypsinized layers were calculated using trypsnized, sliced and H\&E-stained spheroids. Moreover, the distances of neighboring cells were measured and the size of the cells in the viable region and in the core was calculated. Furthermore, the volume of each trypsinized layer and the number of cells within each layer was calculated. The survival curves for cells from the various layers showed that there are subpopulations of cells having different radiosensitivities within each of the outer layers. For these outer layers the most radiosensitive subpopulation showed in situ repair while the most radioresistant seemed not to show it. The core of the spheroids, with the lowest oxygen concentration also showed repair effects for both doses tested. An approximate OER graph was plotted against the surviving fraction for the necrotic cores of the spheroids for 0h and 18h after irradiation. The OER for the core trypsinized 0h after irradiation showed a strong dependence on the relative biological effect with the OER decreasing with decreasing iso-effect, while the core trypsinized 18h after irradiation showed only weak dependence on the effect level.