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The purpose of this study was to compare in vitro the canal and isthmus debridement of manual-dynamic, passive ultrasonic, and laser-activated irrigation with an Er:YAG laser in mesial roots of human mandibular molars.Fifty extracted mandibular molars with an isthmus were embedded in resin and sectioned axially 4 mm from the apex. The teeth were reassembled with guide pins and bolts, and the mesial canals were instrumented up to a ProTaper F2 rotary file (Dentsply Maillefer, Ballaigues, Switzerland). Teeth were randomly assigned to the following irrigant activation groups (n = 10): conventional needle irrigation (NI), manual-dynamic irrigation with a ProTaper F2 gutta-percha cone, ultrasonically activated irrigation using a size 20 Irrisafe (Satelec Acteon, Mérignac, France), and laser-activated irrigation (LAI) with an Er:YAG laser and a conical 400-μm fiber tip in the canal entrance or a 600-μm tip over the canal entrance. Root cross-sectional images were taken before and after final irrigation, and the area occupied by debris in the main canal and the isthmus was determined using image analysis software. Differences in debris before and after activation were statistically compared within and across groups.Significant reductions in debris levels were observed in all groups, except for NI and manual-dynamic irrigation (canal only). None of the methods rendered the canal systems debris free. In the canal, LAI with an Er:YAG laser and a 600-μm tip over the canal entrance removed significantly more debris than NI. In the isthmus, LAI with an Er:YAG laser and a conical 400-μm fiber tip in the canal entrance removed significantly more debris than NI.Within the limitations of this in vitro study, canal and isthmus cleanliness significantly improved after irrigant activation.