The aim of this study was to determine the extent of calcium removal on root canal dentin after 17% EDTA, 17% EGTA, 15% EDTAC, and 1% tetracycline-HCl treatment; with or without subsequent use of 2.5% NaOCl. Extracted single-rooted human teeth were bisected longitudinally and the root halves (n = 100) were isolated with nail varnish, leaving the root canal exposed. The samples were immersed in the test solutions for 1 and 5 minutes, after which the amount of calcium ion (Ca2+) release into the solutions was determined by flame photometry. Regardless of treatment time, all single (treatment solution only) and combined (treatment solution with subsequent NaOCl application) irrigation regimens removed significantly more Ca2+ than control treatment (distilled water). Compared with other groups, treatment with 17% EDTA and 17% EDTA + 2.5% NaOCl resulted in the maximum amount of Ca2+ removal from root canal dentin (p < 0.05). All combined-treatment groups except 17% EGTA + 2.5% NaOCl removed significantly more Ca2+ than their single-treatment versions (p < 0.05). Within each test group, extending the treatment time to 5 minutes resulted in significantly more Ca2+ removal (p < 0.05).