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The purpose of this study was to evaluate the effect of single and combined use of ethylenediamine tetra acetic acid (EDTA), ethylene glycol bis [b-aminoethylether] N,N,N′,N′-tetraacetic acid (EGTA), EDTA plus Cetavlon (EDTAC), tetracycline-HCl, and NaOCl on the microhardness of root canal dentin.The crowns of 30 single-rooted human teeth were discarded at the cementoenamel junction and the roots were bisected longitudinally to obtain root halves (N = 60). The specimens were embedded in autopolymerizing acrylic resin, leaving the root canal dentin exposed. Dentin surfaces were prepared for microhardness test by grinding and polishing. The reference microhardness values of untreated specimens were recorded using a Vicker's microhardness tester at the apical, midroot, and cervical levels of the root canal. Thereafter, the specimens treated with single (test solution only) or combined (test solution, followed by 2.5% NaOCl) versions of the irrigants for 5 minutes. Posttreatment microhardness values were obtained as with initial ones. Statistical comparisons between the test groups and among single and combined treatments were carried out using 2-way ANOVA with repeated measures (P =.05). Comparisons within each group with respect to application regions were made with Friedman's nonparametric 2-way analysis of variance at the same level of significance.All treatment regimens except distilled water significantly decreased the microhardness of the root canal dentin (P < .05). The single and combined use of EDTA decreased the microhardness of the root canal dentin significantly more than all other treatment regimens (P < .05). Compared with their single-treatment versions, all combined treatment regimens decreased the mean microhardness values significantly (P < .05). A comparison of single and combined treatment regimens revealed significant decreases only for EDTA and EDTA + NaOCl in the coronal region and for EDTAC and EDTAC + NaOCl in the apical and middle regions of the root canal (P < .05).The use of EDTA alone or prior to NaOCl resulted in the maximum decrease in dentin microhardness. The softening effect of subsequent NaOCl treatment was both material and region dependent. However, for combined treatment regimens, subsequent use of NaOCl levels the statistical differences between the regional microhardness values obtained after treatment with EGTA, EDTAC, and tetracycline-HCl.