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Introduction: The study evaluated the efficacy of subsonic agitation of sodium hypochlorite (NaOCl) in reducing bacterial load in the root canal. Methods: Root canals of 112 extracted human single-root teeth were preflared using K-Flexofiles (Dentsply Maillefer, Ballaigues, Switzerland) up to #20 and then shaped using ProTaper S1-S2-F1-F2-F3 (Dentsply Maillefer) at the working length. Irrigation was performed with 33 mL of 5% NaOCl, alternating with 10 mL of 10% EDTA. After ethylene oxide sterilization, the root canals were infected with 30 m Lo fEnterococcus faecalis culture and randomly assigned to four groups (n = 25) of different irrigation regimens plus positive and negative controls. Irrigation was performed with 2 mL of 5% NaOCl. In the NaOCl 15 group, the irrigant was left in place for 15 seconds, and in the NaOCl 30 group it was left in place for 30 seconds. In the EndoActivator (EA;Dentsply Tulsa DentalSpecialties, Tulsa,OK) 15and EA 30 groups, NaOCl was subsonically agitated with EA for 15 and 30 seconds, respectively. The residual bacterial count was then evaluated. Differences among groups were analyzed with one-way analysis of variance and the post hoc Bonferroni test (p < 0.05). Results: A statistically significant difference was evidenced among groups (F3 = 9.01, p < 0.001). The standard irrigation groups (NaOCl 15 and 30) showed higher microbial counts than the EA 30 group (p < 0.05). Conclusion: Thirty seconds of NaOCl subsonic agitation with EndoActivator appears to be slightly more effective in reducing bacterial load in the root canal compared with NaOCl irrigation alone. (J Endod 2010;36:1358‐ 1360)