INTRODUCTION: Hard-tissue debris accumulation is a potential side effect of root canal instrumentation that has not been systematically investigated. In the current communication, a method to assess this debris using high-resolution microcomputed tomography (microCT) is presented. METHODS: Based on prescans, mandibular molars with joining mesial root canals and isthmuses between these were selected (n = 6). The mean volume filled with apparent hard-tissue debris after instrumentation without irrigation was calculated over 2 mm of the mesial canal system by multiplying the voxel volume with the number of voxels representing acquired radiopaque material. Backscattered electron imaging was used to compare the calcium-phosphorus content of this material with that of the root dentin in the same specimen. RESULTS: Backscatter scans showed that the accumulated debris viewed in the microCT scans was consistent with root dentin. In the selected canal segments, 29.2% +/- 14.5% of the original canal volume was filled with accumulated debris, which represented a significant change from the preoperative scan (p < 0.01, one-sample t test against zero). Three-dimensional reconstructions of the microCT images visualized the accumulated hard-tissue debris in the whole canal system. CONCLUSIONS: The current method appears suitable to quantitatively compare different instrumenting/irrigating regimens on dentin debris accumulation.