Instrumentation methods currently used in endodontic therapy create a residual layer composed of organic and inorganic material. There is no universal consensus regarding the ideal method for removing this smear layer and exposing the dentinary tubules, necessary for the adaptation of root canal filling. In general, chelating solutions are used but the ideal solution and application time remain unknown. In the present study the chelating power of EDTA, EDTAC and 1, 5 and 10% citric acid (CA) on radicular dentine was analyzed. Co-site digital optical microscopy was used to capture images of several fields of each sample after acid etching for 5 experimental times (15-300 s). An image processing and analysis sequence measured tens of images, providing data of area fraction and size for thousands of tubules over time. Thus, it was possible to follow the phenomenon and quantitatively analyze the effect of the various substances. The CA solutions showed the greatest chelating power. EDTA and CA 1% showed similar effect after 300 s. EDTAC was the least effective. Equivalent experiments employing Environmental Scanning Electron Microscopy provided similar results, complemented by a better view of dentine surface roughness. CA 10% caused severe erosion of the surface