Purpose: To study the influence of tumor fibroblasts on radiosensitivity and stem cell fraction of tumor cells in squamous cell carcinoma megacolonies by determining colony cure and clonogen survival, Methods and Materials: Murine squamous cell carcinoma cells (AT478c) grown as flat but multilayered megacolonies were co-cultured with pre-irradiated tumor fibroblasts derived from the same carcinoma, and irradiated with 1, 2, 4, or 8 fractions. Recurrent clones and their growth pattern in situ were recorded. From megacolony cure data and clonogen survival data, the clonogen number and the parameters of cellular radiosensitivity were calculated, Results: The curability of the co-cultured megacolonies, as determined by TCD50 values, was significantly increased compared to the megacolonies without fibroblasts (p < 0.01). Both the megacolony cure and clonogen survival data suggested a decrease of the clonogen fraction in the so-cultured megacolonies, Conclusion: The presence of tumor fibroblasts increases megacolony radiosensitivity. This is due to a decrease in the fraction of clonogens in the tumor megacolony, apparently caused by a downregulation of the stem cell fraction of the tumor cells.