The aim of this study was to evaluate the influence of different activation protocols for chelating agents used after chemo-mechanical preparation (CMP), for smear layer (SL) removal. Forty-five single-rooted human premolars with straight canals and fully formed apex were selected. The specimens were randomly divided into three groups depending on the chelating agent used for smear layer removal: distilled water (DW, control group); 17% ethylenediaminetetraacetic acid (EDTA); and 10% citric acid (CA). Each group was further divided into three subgroups according to the activation protocol used: no-activation (NA), manual dynamic activation (MDA), or sonic activation (SA). After CMP, all specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy (SEM). Two calibrated evaluators attributed scores to each specimen. The differences between activation protocols were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between each root canal third. When chelating agents were activated, either by MDA or SA, it was obtained the best cleaning results with no significant difference between EDTA and CA (P > 0.05). Sonic activation showed the best results when root canal thirds were analyzed, in comparison to MDA and NA groups (P < 0.05). The activation of chelating agents, independent of the protocol used, benefits smear layer removal from root canals. © 2013 Wiley Periodicals, Inc.