AIM: To evaluate the effect of different root canal irrigating regimes on dentine penetration of Patent Blue dye. METHODOLOGY: Eighty extracted single-rooted human mandibular premolar teeth with narrow root canals were prepared using ProFile instruments. After each instrument, canals were irrigated with 1% sodium hypochlorite. Subsequently, teeth were randomly assigned to receive a 10 mL rinse of aqueous 17% (w/v) ethylenediaminetetraacetic acid or tap water for 2 or 10 min, followed by a final rinse with a 2% Patent Blue dye solution for 2 or 10 min (eight groups, n = 10 teeth per group). Teeth were then horizontally sectioned 3, 6 and 9 mm from the apex. Sections were digitally photographed and dye penetration was calculated as percentage of total dentine area using NIH Image J. Values were compared using one-way anova and Bonferroni correction with the alpha-type error set at <0.05. Representative tooth sections from all groups were further analysed using scanning electron microscopy. RESULTS: No significant impact of irrigating protocols on dye penetration was found. Dye penetration was significantly (P < 0.001) greater in the coronal than middle, and in middle than in apical root thirds. When observed microscopically, irrigant penetration was independent of the presence of a smear layer, but was rather a function of tubular sclerosis. CONCLUSIONS: Tubular sclerosis, a physiological phenomenon that starts in the third decade of life in the apical root region and advances coronally with age, was the main factor influencing penetrability of root dentine.