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Objective: The aim of this study was to compare the effectiveness of five intracanal agitation techniques on the penetration of irrigant and sealer into dentinal tubules. Background data: Intracanal agitation techniques could promote chemomechanical debridement and the sealing of root canals during endodontic treatment. However, there is limited evidence for the agitation effect of Nd:YAP laser. Materials and methods: Human maxillary premolars with single straight canals (N = 60) were prepared with ProTaper Next® NiTi rotary files (Dentsply Maillefer) up to X4 (ISO 40 size). Rhodamine B-labeled sodium hypochlorite was used for final irrigation together with the conventional syringe (control), sonic, ultrasonic, Nd:YAP laser, or V-Clean™ endodontic agitation system. All canals were obturated with gutta-percha and fluorescein isothiocyanate-labeled AH Plus sealer. Transverse sections were obtained at 2, 5, and 8 mm from the apex and observed under confocal laser scanning microscopy. Maximum penetration depth and penetration percentage of both irrigant and sealer were recorded. Kruskal-Wallis and Mann-Whitney tests were performed for multiple comparisons. The Spearman coefficient was calculated to confirm correlations between irrigant and sealer penetration. Results: Laser agitation attained the most irrigant and sealer penetration depth and penetration percentage (p < 0.05). Sonic and ultrasonic agitation attained significantly more penetration percentage than the control group at 2 mm from the apex (p < 0.05). Patterns of irrigant and sealer penetration correlated significantly for all agitation techniques (p < 0.001). Conclusions: Nd:YAP laser was superior to other agitation techniques in dentinal tubule penetration of irrigant and sealer at one or more sectioned levels from the apex.