The major limitation to reaching a curative radiation dose in radioresistant tumors such as malignant gliomas is the high sensitivity to radiation and subsequent damage of the surrounding normal tissues. Novel dose delivery methods such as minibeam radiation therapy (MBRT) may help to overcome this limitation. MBRT utilizes a combination of spatial fractionation of the dose and submillimetric (600 μm) field sizes with an array (“comb”) of parallel thin beams (“teeth”). The dose profiles in MBRT consist of peaks and valleys. In contrast, the seamless irradiations of the several squared centimeter field sizes employed in standard radiotherapy result in homogeneous dose distributions (and consequently, flat dose profiles). The innovative dose delivery methods employed in MBRT, unlike standard radiation therapy, have demonstrated remarkable normal tissue sparing. In this pilot work, we investigated the tolerance of the rat brain after whole-brain MBRT irradiation. A dose escalation was used to study the tissue response as a function of dose, so that a threshold could be established: doses as high as 100 Gy in one fraction were still well tolerated by the rat brain. This finding suggests that MBRT may be used to deliver higher and potentially curative radiation doses in clinical practice.