Aim: To evaluate and compare the ability of smear layer removal and opening of dentinal tubules using 5.25% sodium hypochlorite followed by 17% EDTA .To Evaluate and compare the ability of smear layer removal and opening of dentinal tubules using 5.25% sodium hypochlorite followed by 10% citric acid.Materials and Methods: The roots included in this study were mechanically prepared using crown-down technique. The roots were randomly divided to Group 1: final irrigation sequence was 3 ml of 5.25% NaOCl for 1 minute followed by 3 ml of distilled water for 1 minute. Group 2: final irrigation sequence was 3 ml of 5.25% NaOCl for 1 minute followed by 3 ml of 17% EDTA for 1 minute. Group 3: final irrigation sequence was 3 ml of 5.25% NaOCl for 1 minute followed by 3 ml of 10% citric acid for 1 minute. Root canal dentin was examined using an environmental scanning electron microscope (Quanta FEG 250 (NRC)). Microphotographs were obtained from each specimen at a magnification of 2000X. Evaluation of the smear layer removal and opening of dentinal tubules was achieved using computerized image analysis software (Leica QWin 500), which demonstrated the area of total opened dentinal tubules in relation to the area representing the total dentinal surface area of each microphotograph which was then expressed in percentage.Results: The results of this study revealed typical smear layer appearance, showing cracked, thick and amorphous smear layer covering all different root canal levels showing no exposed opened dentinal tubules in roots irrigated by NaOCl followed by distilled water (Group 1), therefore this group was excluded from the statistical analysis. However, Group 2 and 3 showed different degree of smear layer removal and opening of dentinal tubules, revealing statistically significant difference between them regarding all different root canal levels, showing the EDTA group (Group 2) was higher than the citric acid group (Group 3).Conclusion: This study concluded that 17% EDTA was more effective in smear layer removal and opening dentinal tubules than 10% citric acid in all root canal levels (coronal, middle, and apical). The coronal third of both tested groups showed the cleanest level, while the apical third showed the least cleanliness. NaOCl followed by distilled water did not remove the smear layer and open dentinal tubules.