Objectives: The aim of this study was to test the hypothesis, that the effectiveness of irrigation in removing smear layer in the apical third of root canal system is dependent on the depth of placement of the irrigation needle into the root canal and the enlargement size of the canal. Materials and Methods: Eighty sound human lower incisors were divided into eight groups according to the enlargement size (#25, #30, #35 and #40) and the needle penetration depth (3 mm from working length, WL-3 mm and 9 mm from working length, WL-9 mm). Each canal was enlarged to working length with Profile.06 Rotary Ni-Ti files and irrigated with 5.25% NaOCl. Then, each canal received a final irrigation with 3 mL of 3% EDTA for 4 min, followed by 5 mL of 5.25% NaOCl at different level (WL-3 mm and WL-9 mm) from working length. Each specimen was prepared for the scanning electron microscope (SEM). Photographs of the 3mm area from the apical constriction of each canal with a magnification of ${ imes}250$, ${ imes}500$, ${ imes}1,000$, ${ imes}2,500$ were taken for the final evaluation. Results: Removal of smear layer in WL-3 mm group showed a significantly different effect when the canal was enlarged to larger than #30. There was a significant difference in removing apical smear layer between the needle penetration depth of WL-3 mm and WL-9 mm. Conclusions: Removal of smear layer from the apical portion of root canals was effectively accomplished with apical instrumentation to #35/40 06 taper file and 3 mm needle penetration from the working length.