We have conducted the first in-vivo experiments in pencilbeam irradiation, a new synchrotron radiation technique based on the principle of microbeam irradiation, a concept of spatially fractionated high-dose irradiation. In an animal model of adult C57 BL/6J mice we have determined technical and physiological limitations with the present technical setup of the technique. Fifty-eight animals were distributed in eleven experimental groups, ten groups receiving whole brain radiotherapy with arrays of 50 µm wide beams. We have tested peak doses ranging between 172 Gy and 2,298 Gy at 3 mm depth. Animals in five groups received whole brain radiotherapy with a center-to-center (ctc) distance of 200 µm and a peak-to-valley ratio (PVDR) of ∼ 100, in the other five groups the ctc was 400 µm (PVDR ∼ 400). Motor and memory abilities were assessed during a six months observation period following irradiation. The lower dose limit, determined by the technical equipment, was at 172 Gy. The LD50 was about 1,164 Gy for a ctc of 200 µm and higher than 2,298 Gy for a ctc of 400 µm. Age-dependent loss in motor and memory performance was seen in all groups. Better overall performance (close to that of healthy controls) was seen in the groups irradiated with a ctc of 400 µm.