Abstract Objectives. The goal of this study is to determine if the smear layer affects the passage of bacteria through or around obturating material as evidenced by penetration of bacteria through and out the canal. Specifically, this study focused on determining the effect of the smear layer on the magnitude of bacterial penetration through the apical foramen. Methods. Thirty extracted, maxillary central or lateral incisors were collected. Teeth were randomly assigned (10 teeth per group) to three groups: (1) smear layer removed, (2) smear layer present, and (3) negative control. Canal preparation and obturation using lateral condensation, gutta percha, and AH 26 sealer was performed on all of the teeth. Removal of the smear layer was accomplished by rinsing with 17% EDTA. The model systems consisted of an upper chamber attached to the cemento-enamel junction and a lower chamber at the apices of the teeth. Standardized bacterial suspensions containing Fusobacterium nucleatum, Campylobacter rectus, and Peptostreptococcus micros were inoculated into the upper chambers. Models were incubated anaerobically at 37 °C. At various times over a 60-day period, samples were taken from the lower chamber and spiral-plated on selective-differential media to determine numbers and types of bacteria. Results. Leakage results were as follows: (1) smear layer present—6/10 leaked; (2) smear layer removed—0/10 leaked; (3) negative control—0/10 leaked. Profiles of bacterial leakage were similar among the groups. F. nucleatum was the predominant microorganism. Conclusions. This study indicated that removal of the smear layer reduced the leakage of bacteria through the root canal system.