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Objectives: This study using contralateral teeth from human cadavers assessed the amount of unprepared wall surface areas (USA), accumulated hard tissue debris (AHTD), and dentinal crack formation after root canal preparation using either reciprocating or rotary instruments., Materials and Methods: Incisors with a single root canal from 11 human mandibles had their root canals prepared with Reciproc or Mtwo. Each contralateral tooth was treated with one of the test instruments in order to create matched pairs. Micro-CT scans were taken before and after preparation to instrument sizes 40 and 50 in both groups. USA and AHTD were evaluated in the full canal length or the apical portion only. Crack formation was also evaluated in the full root length., Results: After size 40, no significant differences in USA and AHTD were observed in the full canal length (P > .05). Separate analysis of the apical 4-mm canal revealed no difference in AHTD, but significantly more USA with Mtwo than Reciproc (P < .05). After size 50, the amount of USA was similar between groups both in the full canal and in the apical canal (P > .05). More AHTD were observed in the full canal after using Mtwo size 50 (P < .05), but no difference occurred in the apical canal only (P > .05). Intragroup analyses showed a significant decrease of USA and a significant increase of AHTD when preparation size increased from 40 to 50 (P < .05). In teeth without any pre-existing detectable dentinal defect (n = 38), crack formation occurred in 4 teeth, 2 from each preparation system., Clinical Relevance: Reciproc instrument size 40 resulted in more prepared areas in the apical canal than Mtwo instrument of the same size. However, the amount of AHTD did not differ between them at this file size. Canal enlargement to size 50 prepared more walls but created more debris. Dentinal defects were produced after preparation with both systems., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)