The purpose of this in vitro study was to evaluate what influence an interappointment dressing with calcium hydroxide might have on the ability to subsequently obturate simulated lateral canals. Twenty extracted single-rooted human teeth were instrumented at the working length to a #35 file before creating three simulated lateral canals on the mesial and distal surfaces of the root, one in each third, using a #15 engine reamer. After enlarging the root canals to a #45 file, the teeth were randomly divided into two groups of 10. In both groups AH26 was used as the sealer. In group A, the obturation was performed immediately using Ultrafil. In group B, root canals were filled with calcium hydroxide and incubated for 7 days. The teeth were then reinstrumented, irrigated with 2.5% sodium hypochlorite solution, dried with paper points, and obturated as in group A. Teeth in group A showed a larger number of filled lateral canals compared with those in group B. Statistical significant differences were observed when comparing the same third in the two groups (apical third p < 0.05, middle third p < 0.01, and coronal third p < 0.01). No differences were found among the different thirds in the same group (p > 0.05).