The purpose of this study was to biochemically compare the decalcifying effects of 1% EDTA (pH 7.4), 1% EGTA (pH 7.4), 1% CDTA (pH 7.4), 1% citric acid solutions (pH 1.0 and 7.4) and saline solution (control) on root dentin. Forty-eight single-rooted teeth were used in this study. The canals were instrumented by the step-back technique and the roots were randomly divided into six equal experimental groups (n = 8) according to the irrigating agent tested. A total of 30 microL of each solution was pipetted into the root canal and allowed to set undisturbed for 5 minutes. After this time, 15 microL of the solutions were removed from each canal using a Hamilton syringe and placed in a container with 5 mL of deionised water. The microg/mL concentration of calcium ion (Ca2+) extracted from the root canal samples was determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Data were analysed by means of the Kruskal-Wallis and Mood's median tests. Citric acid solution at pH 1.0 removed more calcium than at pH 7.4 and than the other chelating solutions tested (p < 0.05). No differences were observed between EDTA and EGTA. Both EDTA and EGTA removed significantly more calcium than CDTA and citric acid at pH 7.4 (p < 0.05). There were no differences between citric acid at pH 7.4 and saline solution, which had the least efficacy for Ca2+ extraction (p > 0.05). These results indicate that citric acid at pH 1.0 is a good alternative as an irrigating solution to remove the smear layer and facilitate the biomechanical procedures.