The aim of this study was to compare in vitro reduction of Enterococcus faecalis by using three agitation methods. Fifty-five mandibular premolar teeth were used. The root canals were prepared by using ProTaper instruments to size F4. Fresh bacterial suspensions were inoculated into each canal and the teeth were incubated at 37 degrees C under aerobic conditions for 3 weeks. The specimens in negative control group were not contaminated and the teeth in positive control receive no further instrumentation or irrigation after contamination. The contaminated teeth were divided into three experimental groups and two control groups. The experimental groups were as follows according to agitation of NaOCl irrigation solution; group 1, the brush-covered NaviTip FX needle; group 2, EndoVac apical negative pressure irrigation system; and group 3, self-adjusting file (SAF). Following the final irrigation, samples obtained with sterile #40 H-file from teeth and the file used were placed into sterile microcentrifuge tubes. After the incubation period, bacterial colony numbers were noted as CFU/ml. Three teeths were randomly selected from each group for scanning electron microscope evaluation. The three groups were compared with the Kruskal-Wallis test. Comparisons between three groups were made with the Mann-Whitney U-test with Bonferroni correction. SAF was significantly effective in elimination of E. faecalis when compared to EndoVac (p < 0.05). There was no difference between NaviTip FX needle and other two methods (p > 0.05). In conclusion, agitation of NaOCl with SAF for 2 min may be an effective alternative approach. (c) 2014 Wiley Periodicals, Inc.