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The purpose of this study was to evaluate the ultramorphological and chemical changes in Er, Cr:YSGG laser-treated radicular dentin in comparison with two different irrigation regimes. Extracted human premolars were assigned into three experimental groups. Group 1: After each file size, root canals were irrigated with 5.25% NaOCL, followed by a final irrigation with 5.25% NaOCL. Group 2: At each file size, biomechanical preparation was performed in conjunction with Rc-Prep, followed by 5.25% NaOCL irrigation; and 5.25% NaOCL for the final flush. Group 3: 5.25% NaOCL was used after each file size, followed by Er, Cr:YSGG laser irradiation; and a final flush with 5.25% NaOCL. Scanning electron microscopic (SEM) evaluations revealed failure of smear removal in NaOCL-irrigated specimens. RC-Prep + NaOCL-treated dentin revealed moderate-to-total presence of the smear layer with distinct areas of exposed collagen. Er, Cr:YSGG laser irradiation of radicular dentin also resulted in partial or total removal of the smear associated with a few small regions of thermal injury, including carbonization and partial melting. Energy dispersive X-ray analysis (SEM-EDX) showed no significant difference between the Ca/P ratios of the test groups, suggesting absence of changes at the molecular level.