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The aims of this in vitro study were to evaluate debris and smear layer elimination capability effectiveness of 5.25% sodium hypochlorite (NaOCl) after calcium hydroxide removal from the root canal and to determine if ethylenediaminetetraacetic acid (EDTA) and citric acid have any additional effects on debris and smear layer removal.All irrigation regimens are associated with ultrasonics. Thirty-six pulpless single-rooted teeth were divided into three groups on the basis of the irrigation regimen, namely 5.25% NaOCl alone, 5.25% NaOCl with 17% EDTA pH 7 (NaOCl-EDTA), and 5.25% NaOCl with 50% citric acid (NaOCl-CA). All root canals were instrumented with the Protaper rotary system (Dentsply-Maillefer, Ballaigues, Switzerland) using a crown-down approach. The apical foramen of each tooth was enlarged to a size 30 with a 0.09 taper. After longitudinal sectioning, the dentin walls were microphotographed using scanning electron microscopy (SEM) at a 1500x magnification. Using a grid and a 5-step scale these microphotographs were scored by three investigators in order to evaluate debris and smear layer residuals. Data were statistically evaluated using Kruskal-Wallis and Mann-Whitney tests.In the coronal third the lowest score obtained with NaOCl-CA was similar to NaOCl-EDTA. When using NaOCl-EDTA, mean debris scores were similar to the control group (NaOCl) and lower than NaOCl-CA in the middle and apical thirds. Regarding the mean smear layer score, NaOCl-CA was the lowest in the coronal, middle, and apical thirds with a statistically significant difference (p0.001).When the total surface of the root was evaluated, NaOCl-EDTA was superior in removing debris, but the association of NaOCl-CA enabled the most effective removal of the smear layer.