The aim was to assess the impact of irrigation protocols ended with ethanol or chlorhexidine on AH Plus penetration into dentinal tubules. 45 root canals were prepared to ISO 40/04, divided into three groups and irrigated with three protocols: Group 1 (control): 5.25% NaOCl; Group 2: 40% CA (citric acid), 5.25% NaOCl, 40% CA, water, ethanol; Group 3: 40% CA, 5.25% NaOCl, 40% CA, water, 2% chlorhexidine. Canals were filled using vertical condensation technique with gutta-percha and fluorescein-stained AH Plus sealer. After 72 h, 1 mm thick cross-sections were cut at 2, 5, 8 mm from the apex. Confocal laser microscope was used to measure the sealer penetration into dentinal tubules. Mean depth of sealer penetration (in micrometres) was 107, 131, 170 (Group 1); 146, 233, 317 (Group 2); 185, 301, 542 (Group 3); in apical, middle and coronal parts, respectively. Irrigation protocol ended with chlorhexidine resulted in the deeper sealer penetration compared with alcohol.