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To evaluate the ability of electrophoretically activated calcium hydroxide (CH) to eliminate bacteria in dentinal tubules. In an in vitro model of dentinal tubule infection, 18 cylindrical root specimens prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 21 days. In 12 specimens, CH paste was placed in the root canals for 7 days. In six of these, an electrophoretic current (10 mA per 10 min), using two electrodes, was applied after placing the medicament in the canal. Powder dentin samples obtained from within the canal lumina using ISO 025, 027, 029, 031 and 033 burs were examined for the presence of vital bacteria by inoculating agar plates and counting colony forming units. ANOVA with repeated measures was used to analyze results. A significant difference was found between experimental groups and the positive control group. CH and electrophoretically activated CH significantly (P < 0.001) reduced bacterial viabilities in dentinal tubules to a depth of 200 μm. Treatment with electrophoresis was significantly (P < 0.001) more effective than pure CH in depths of 200–500 μm. Specimens treated with electrophoretically activated CH showed no viable bacteria in dentinal tubules to a depth of 500 μm from the root canal space within 7 days. The time required for treatment of pulpal infection root resorption may be decreased, thus minimizing the risk of coronal fractures in young patients with traumatized teeth.