INTRODUCTION: The purpose of this in vitro study was to assess the apical microleakage of AH26 sealer when three different root canal irrigant regimens were used. MATERIALS AND METHODS: Eighty single-rooted human teeth were randomly divided into three experimental (n=20) and two control groups (n=10). NaOCl was used as irrigant during instrumentation, and apical patency was ensured in all teeth. Final irrigation was implemented as follow: group A- 17% EDTA + 5.25% NaOCl, Group B- 7% citric acid + 5.25% NaOCl, and group C- 20% citric acid + 5.25% NaOCl. The experimental and negative control groups were obturated by laterally condensed gutta-percha with AH26 sealer. The positive control group was obturated without sealer. The teeth were stored in 100% humidity and 37ºC for 48 hours. In the experimental groups and positive control group, the root surfaces except for the apical 2 mm were covered with nail polish and sticky wax. In the negative control group, the roots were completely covered. The samples were then immersed in 2% methylene blue dye for 72 hours at 37ºC. After that the roots were sectioned longitudinally and the dye penetration was measured. The results were statistically analyzed by One-way Variance and Post Hoc Tukey tests. RESULTS: Statistically significant difference was found between groups (P<0.05). Group C showed the least (1.072 mm) and group A showed the most (2.072 mm) amount of dye penetration. CONCLUSION: When a resin-based sealer is used for the obturation of the root canal system, it is better to use a citric acid irrigant instead of EDTA to remove the smear layer and to improve the apical seal.