Aim To evaluate the accumulation of hard-tissue debris when using the Self-adjusting File (SAF) system in mesial roots of mandibular molars with isthmuses and to compare it with that occurring when the ProTaper file system was used. Methodology Forty extracted human mandibular molars with joining mesial root canals and an isthmus between the two canals were randomly assigned to two experimental groups and scanned using micro-computed tomography. Root canals in the control group (N=20) were instrumented using the ProTaper rotary system until F3. Irrigation with 1mL of 3% NaOCl solution was applied after each instrument. Root canals in the experimental group (N=20) were prepared using the SAF for 4min, with continuous irrigation (3% NaOCl, 4mLmin -1). After rescanning, canals in both groups were further irrigated with 5mL of 17% EDTA for 2min. After final scanning, the per cent value of total canal system volume filled with hard-tissue debris was calculated. Statistical analysis was performed using Mann-Whitney U-test; the alpha-type error was set at 1%. Results Instrumentation of the root canals with ProTaper left 10.1% (IQR 5.2) of the total canal system volume filled with hard-tissue debris while preparation with the SAF left 1.7% (IQR 1.6). This difference was highly significant (P<0.0001). After subsequent EDTA irrigation, these values were significantly reduced (P<0.01) to 7.9% (IQR 4.1) and 1.3% (IQR 0.8) in the ProTaper and SAF groups, respectively. Conclusion Preparation with the SAF system resulted in less hard-tissue debris accumulation in isthmus-containing root canal systems compared with instrumentation with ProTaper rotary files. © 2011 International Endodontic Journal.