In the 70's it was discovered that after instrumentation of the root canal there was the formation of a smear layer, attached to the walls of the canal. This layer is composed of organic and inorganic material, pulp tissue and bacteria, and contributes to the obliteration of the dentine tubules. The removal of this layer improves the adaptation of the filling material to the dentine walls, eliminates bacteria, cleans and enlarges the tubules. Smear layer removal is achieved through the use of chelators but the ideal substance and application time remain unknown. Chelators act through the removal of Calcium from the dentine microstructure. In the present study, the chelating power of MTAD, HEBP and EDTA on coronal dentine was analyzed through the measurement of the area fraction of open tubules (AreaP) and of the chelated Calcium mass (MNC(Ca)). Three hypotheses were proposed and tested: There is no difference between chelators regarding AreaP (H1) and regarding MNC(Ca) (H2); there is correlation between AreaP and MNC(Ca) (H3). Images of several fields of each sample, for different chelation times, were acquired through Co-Site Optical Microscopy (CSOM). An image processing and analysis sequence was employed to measure AreaP versus time, for each chelator. This analysis was qualitatively complemented by Scanning Electron Microscopy (SEM). Measurements of MNC(Ca) were obtained by Atomic Absorption Spectroscopy (AA) from the chelator solutions used during the CSOM experiments. H1 was denied, with MTAD found to be the most powerful chelator while HEBP was the weakest. SEM results corroborate these findings. AA results showed wide dispersion but indicate that H2 is false. H3 was rejected.