Background The heating of chelating agents such as EDTA increases dentin wettability by decreasing surface tension. However, the calcium ion release effect of preheated chelating agents in instrumented root canals has not yet been mentioned. In this study, it was aimed to evaluate the number of calcium ions removed by the pre-heated chelating agents from the root canals. Material and Methods After 51 bovine teeth were instrumented, three of them were separated as negative controls and the remaining teeth were divided into six groups according to the temperature of the solution (at 22 or 37ºC): EDTA-22, CITRIC-22, QMix-22, EDTA-37, CITRIC-37 and QMix-37. Following irrigation, calcium ion levels were determined by atomic absorption spectrophotometer in chelating agents collected from the root canals. Results QMix solution eliminated significantly more calcium ions than other chelating agents at different temperatures ( p < 0.05). Regardless of the heating, QMix and 17% EDTA were significantly superior to 40% Citric acid ( p < 0.05) while no significant difference was detected between QMix and 17% EDTA groups ( p < 0.05). Heating all chelating agents did not significantly increase their ability to remove calcium ions from pre-instrumented root canals ( P < 0.05). In the SEM examination, it was observed that the smear layer was removed from the middle third of the roots, except for the negative control group. Conclusions Temperature changes have shown that these agents do not increase the ability of the smear layer to dissolve the inorganic structure. QMix at different temperatures may be recommended to use as the final chelating agent. Key words: EDTA, citric acid, QMix, calcium ions, temperature.