Introduction: The purpose of this study was to evaluate the effect of calcium hydroxide (Ca[OH](2)) dressing on the dentinal tubule penetration of epoxy resin based sealer (AH 26; Dentsply Maillefer, Ballaigues, Switzerland) and tricalcium silicate based sealer (BioRoot RCS; Septodont, Saint Maurdes Fosses, France). Methods: Fifty-two single -rooted mandibular premolars were used. Four samples were assigned as the positive control. Twenty-four samples received Ca(OH)(2) labeled with rhodamine B, whereas the rest did not. Ca(OH)(2) was removed with passive ultrasonic activation and copious irrigation 2 weeks later. Samples were further subdivided into 2 groups, and root canal fillings were performed with a single ProTaper F4 gutta-percha cone (Dentsply Maillefer) combined with 1 of the tested sealers labeled with fluorescein green. After 2 weeks, samples were transversely sectioned at the apical, middle, and coronal levels. The penetration depth and percentage were evaluated via imaging software. Statistical analysis was performed using KruskalWallis, Siegel Castellan post hoc, and Mann -Whitney U tests at P = .05. Results: The mean dentinal tubule penetration depth and percentage values were lowest in the apical third for both sealers. BioRoot RCS showed higher penetrability in all thirds compared with AH 26 (P < .05) despite Ca(OH)(2) dressing remnants (P < .05). Ca(OH)(2) placement resulted in a shorter dentinal tubule penetration depth with BioRoot RCS statistically in the middle and coronal thirds (P < .05), whereas it did not affect the percentage (P > .05). Conclusions: Passive ultrasonic activation and copious irrigation were insufficient in removing Ca(OH)(2) from root canals. BioRoot RCS presented higher dentinal tubule penetration than AH 26 even in the presence of Ca(OH)(2) residues. Ca(OH)(2) remnants decreased both dentinal tubule penetration depth and the percentage of the tested sealers; however, a more drastic effect was observed for AH 26.