PURPOSE: To compare the removal of smear layer and the structure of the hybrid layer formed after the use of EDTA or MTAD solutions when used as a final flush. METHODS: Single-rooted extracted premolars (n=18) were collected and treated for root canal therapy using NaOCl irrigation followed by a final rinse of 17% EDTA, Biopure MTAD, or saline (negative control). The roots were obturated with gutta-percha and a hydrophilic HEMA-containing root canal sealer. The TEM specimens were impregnated with 50% silver nitrate to visualize sealing imperfections and nanoleakage. The structure of the coronal, middle and apical parts of root canal walls was examined using transmission electron microscopy. RESULTS: After NaOCl irrigation, a final rinse with BioPure MTAD or 17% EDTA completely removed the 2 microm-thick smear layer on mechanically instrumented root canal walls. The BioPure MTAD hybrid layer was thicker than the 17% EDTA hybrid layer. Both the BioPure MTAD and EDTA caused a collapse of the dentin matrix structure which impeded sealer infiltration and the formation of high quality hybrid layer bonding. The hybrid layers created in smear layer-covered dentin exhibited less potential for nanoleakage than the MTAD or EDTA hybrid layers.