Objectives:This study comparatively evaluated the efficacy of self-adjusting file (SAF), Endovac, and CanalBrush irrigant agitation protocols in removing calcium hydroxide (Ca(OH)2) from the root canals.Materials and Methods:Sixty extracted human mandibular canine teeth were instrumented with ProTaper rotary instruments to size #40 and dressed with Ca(OH)2. The roots were randomly assigned to four groups according to irrigant agitation protocol used (n = 15). In Group 1: Conventional syringe irrigation (no activation, control); Group 2: Rotary brush agitation (CanalBrush); Group 3: Apical negative pressure irrigation (EndoVac system); and Group 4: Sonic agitation (SAF) were used. Scanning electron microscopic (SEM) evaluation was done for assessment of Ca(OH)2 removal in the coronal and apical thirds. Statistical analysis was performed by Wilcoxon and Kruskal-Wallis tests.Results:There were statistically significant differences among the groups (P = 0.218). A statistically significant difference was seen between the test groups in Ca(OH)2 removal from the apical third of the canal (P < 0.05). In the coronal third, there was no difference between the groups (P > 0.05). The most efficient Ca(OH)2 removal in apical third was recorded in Group 3 (EndoVac) and Group 4 (SAF) (P < 0.05). In Group 4 (sonic agitation), there was no significantly difference between Ca(OH)2 removal in coronal and apical thirds.Conclusions:SAF and EndoVac showed significantly better performance than CanalBrush and conventional syringe irrigation in removing Ca(OH)2 from apical third of the root canals.