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Aim: To use micro-CT to quantitatively evaluate the amount of residual filling material after using several techniques to remove root fillings with and without ultrasonic activation and to analyse the cleanliness of the root canal walls and dentine tubules with scanning electron microscopy (SEM)., Methodology: The root canals of one hundred and eight human mandibular incisors were selected and instrumented with rotary files using the BioRace system up to file size 40, .04 taper. After instrumentation, the teeth were filled using a hybrid technique with gutta-percha and sealer then divided into three groups according to the method used for removing the root filling: G1-Reciproc (using only instrument R50), G2-ProTaper Universal retreatment system and G3-Manual (hand files and Gates-Glidden burs). All groups were divided into two subgroups depending on whether ultrasonic agitation was used with the irrigants. Micro-CT scans were taken before and after removal of the filling material to detect residual material in the canal. After micro-CT analysis, the roots were cut in half, imaged by SEM and scored based on the amount of surface covered by root filling remnants. The data were analysed statistically using a significance level of 5%., Results: All groups had retained material in the root canals after instrumentation. The Reciproc method was associated with less retained material than the ProTaper and Manual methods. Ultrasonic activation significantly reduced the amount of residual root filling in all groups (P < 0.05)., Conclusion: None of the instrumentation methods were able to completely remove root filling material. Ultrasonic activation improved the removal of root filling material in all groups., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)