The purpose of this study was to evaluate the effect of the presence or absence of amorphous, irregular smear layers on the adhesion of Prevotella nigrescens, to the dentin of the root canal by using scanning electron microscopy (SEM). Human incisors extracted within 7 days, with no cavities, no fractures, and no evidence of calcification of the canal, were selected. After cutting the crown portion at the CEJ, root canal preparation was undertaken by using a modified crown-down technique with Profile and Gates Glidden drills. Ten milliliters of physiologic saline solution (groups 1 and 4), 10 ml of 3.5% NaOCl (groups 2 and 5), or 10 ml of NaOCl and 10 ml of 0.5 M EDTA (groups 3 and 6) were used as irrigation solution while preparing the canal. After vertical sectioning and ethylene oxide gas sterilization, samples (groups 1, 2, and 3) were immersed in brain-heart infusion broth with yeast extract, hemin, and menadione, inoculated with P. nigrescens (ATCC 33563), and incubated for 3 h at 37 degrees C. All samples were prepared for and observed with SEM. The data were analyzed by using t test and one-way ANOVA. Smear layer was observed to cover the entire root canal surface after root canal preparation. Smear layer was removed and the entrances of dentinal tubules opened widely after applying 3.5% NaOCl and 0.5 M EDTA. A significantly greater number of bacteria were found to adhere to those teeth in which a smear layer was present (p < 0.05). Given that the smear layer produced during root canal preparation promoted adhesion and colonization of P. nigrescens to the dentin matrix, it might also increase the likelihood of canal reinfection.