To evaluate the effect of smear layer on apical microleakage in extracted root-filled teeth by SEM and glucose penetration model.51 straight single-rooted human teeth were randomly divided into 5 groups, instrumented and irrigated differently by groups. 11 teeth in group A were irrigated with 17% EDTA and 1% NaClO,11 teeth in group B with 1% tetracycline hydrochloride and 1% NaClO,11 teeth in group C with 10% citric acid and 1% NaClO, 9 teeth in group D with 1% NaClO only and 9 teeth in group E with 0.9% NaCl. One tooth in each group was observed under scanning electron microscopy. The other teeth were obturated using lateral condensation technique. Leakage along the root filling was measured by the concentration of glucose in apical reservoir at 1, 2, 4, 7, 10, 15, 20 and 30 days respectively with glucose oxidase method. The data were analyzed with repeated measure ANOVA and SNK-q test using SPSS 16.0 software package.No smear layer on the root canal wall was observed in group A, B and C, the leakage was lower than that in group D and E (P<0.05). The smear layer occurred in quantity in group D, E and their leakage increased. The microleakage was higher in group E than that in group D (P<0.05).Removal of smear layer on the root canal wall could reduce the root apical microleakage.