The aim of this in vitro study was to determine the effect of three different root canal sealers on the bacterial penetration of obturated root canals. Eighty extracted human teeth with straight, single root canals were prepared using the modified double-flared technique with balanced force under copious irrigation until the master apical file was size 40. The teeth were divided randomly into experimental groups (60 teeth) and control groups (20 teeth). Twenty experimental teeth each were obturated by lateral condensation of cold gutta-percha with AH26, Apexit or Tubliseal EWT sealer. In the control groups, 10 teeth were obturated using the same technique with AH26, Apexit or Tubliseal EWT sealer. These teeth were completely sealed to serve as negative controls. The remaining 10 teeth were not obturated and served as positive controls. The root surface of each tooth, except the apical 2 mm, was sealed with nail varnish. The coronal part of each root canal was sealed with the cut end of a polypropylene tube and placed in a glass bottle containing sterile Todd-Hewitt Broth (THB). Aliquots of 0.5 ml of THB were injected into the polypropylene tube and the model system was centrifuged at 168 g. An innoculum of Streptococcus sanguis in THB was added to the tube at 5-day intervals and daily observations were made for bacterial growth in the apical reservoir for 90 days. All positive control teeth showed bacterial penetration within 24 h and negative control teeth remained uncontaminated throughout the test period. Leakage through the experimental teeth varied from 7 to 86 days. There was no statistically significant difference (P > 0.05) in leakage of the obturated canals between AH26, Apexit and Tubliseal EWT sealers.