This study histologically compared the in vivo debridement efficacy of the step-back preparation versus a step-back/ultrasound preparation in the mesial root canals of vital mandibular molars. Group 1 consisted of 17 teeth prepared with a step-back technique using intermittent irrigation with 5.25% sodium hypochlorite. Group 2 consisted of 17 teeth prepared with a step-back technique as in group 1 followed by 3 min of ultrasonic instrumentation per canal utilizing a #15 Endosonic file in an Enac unit set at 3.5. An additional 6 ml/canal of 5.25% sodium hypochlorite was used during the ultrasonic preparation. Eight uninstrumented mandibular molars served as histological controls. Following extraction and histological preparation, 0.2-microns cross-sections from the 1- to 3-mm apical levels of the canal and isthmus were evaluated for percentage of tissue removal using an Olympus CUE-2 Image Analysis System. Factorial analysis of variance indicated canal and isthmus cleanliness values were significantly higher, at all 11 apical levels, with the ultrasonic technique. Sample values at the 1-, 2-, and 3-mm levels for the step-back and step-back/ultrasonic techniques, respectively, were: canal, 64% versus 92%, 81% versus 97%, and 90% versus 99.9%; isthmus, 2% versus 46%, 15% versus 60%, and 16% versus 83%.