Statement of problem: Periapical surgery has been performed to remove irritant factors and seal the end of the root. So, selection of retrograde filling materials which can produce hermetic seal and are easily available with reasonable prices seem to be necessary.Purpose: The aim of this in-vitro study was to compare the sealing ability of Pro Root MTA, Root- MTA and Portland cement.Materials and Method: In this experimental in-vitro study, 40 intact mandibular first premolar teeth were selected and cut from the site of cemento-enamel junction (CEJ). The canals were prepared using step back method and obturated with Gutta-Percha and sealer by lateral condensation technique. The 3 mm of the apical portion of the roots were cut with a 45 angle to the long axis of the teeth. Thirty six teeth were divided into three experimental groups and the 4 remaining teeth were considered as control groups. Cavities randomly with 3 mm depth were prepared in the apical of the canals by a ½ round bar, and in the experimental groups they were filled with pro-root MTA, root MTA and/or Portland cement. The surfaces of the roots were covered by nail polish. After incubations, the teeth were kept in %2 methylen blue solution for 24 hours. Then, the teeth were cut longitudinally and the depths of color penetration were evaluated by a stereo microscope. The data were analyzed using one-way ANOVA and Mann-Whitney U test.Results: In the positive control group, the dye had penetrated the total cavity; in the negative control group, microleakage was not found. There was no significant differences between the Pro Root MTA and Root MTA ( p >0.05), but Pro Root MTA and Root MTA had significant differences with Portland cement ( p <0.05). Pro-root MTA had the lowest apical microleakage followed by root MTA and Portland cement respectively.Conclusion: Considering the results of this study, Portland cement cannot be replaced with Pro-root MTA in retrograde fillings, but using Root MTA as a substitute for Pro Root MTA needs more investigations.Key words: Microleakage, Pro Root MTA, Portland cement, Root MTA, Root canal filling material