The purpose of this study was to evaluate the effect of smear layer on penetration of four root sealers into dentinal tubules. Sixty-four, recently extracted, human maxillary and mandibular incisors were used. Following removal of the crowns, the pulps were extirpated and the root canals instrumented. The teeth were then divided into two groups. The teeth in the control group were irrigated with saline solution and the experimental group treated with EDTA and NaOCl. Each main group was then divided into four sub-groups and the root canals filled with Diaket, N2 Universal, SPAD and Forfenan as recommended by the manufacturers. The teeth were split longitudinally and examined using scanning electron microscopy. A quantitative assessment was made of sealer penetration into the dentinal tubules in the coronal, middle and apical parts of each root canal. It was observed that the smear layer obstructed the penetration of the tubules by the sealers. However, in the experimental group, the penetration into dentinal tubules was better with Diaket, N2 and SPAD, than with Forfenan (P < 0.01). It was concluded that this penetration could be affected by physical and chemical properties of root canal filling materials.