The purpose of the present study was to verity whether different conditions of contact with a dye solution would result in different levels of the impregnation by the marker agent in human radicular dentin. The root canals of 60 upper human canines were immersed in methylene blue dye (0.5%, pH 7.0) for 24 h under six different conditions: passive immersion; pre-agitation for 10 min using an endodontic ultrasound; pre-agitation for 10 min using a cleaning ultrasound; 25-mmHg vacuum for 10 min followed by passive immersion; 30-mmHg and 650-mmHg vacuum for 24 h. The roots were longitudinally sectioned and dye impregnation was assessed by a trained and calibrated examiner, using the Q 550 IW image analyser (Leica Qwin, Cambridge, England). The measurements were analysed with anova and Tukey test. The results revealed no significant difference among the different methods for dye impregnation. In the cervical third, impregnation with 30 mmHg was significantly more effective than with endodontic ultrasound or 25 mmHg. At the apical third, no differences were observed between any of the dye impregnation methods. The comparison of different thirds revealed a statistically significant difference between the cervical and apical and the middle and apical thirds, but not between the cervical and middle thirds.