During mechanical preparation of the post space, the root canal filling may be twisted or vibrated, depending on several factors associated with the preparation technique and quality of filling. Objective: The purpose of this study was to investigate the effect of immediate and delayed post space preparation on the integrity of the apical seal. Material and Methods: Sixty-four extracted human incisors were biomechanically prepared using the step-back technique. Sixty roots were randomly assigned to 6 experimental groups of 10 teeth each and the remaining 4 roots served as positive and negative controls (n=2). The root canals in the different groups were obturated with cold lateral and warm vertical condensation of gutta-percha and one of two sealers (Sealapex and Diaket). Post space was prepared either individually or simultaneously. An insulated copper wire was cut into 10-cm-long pieces. In each canal, one piece was inserted to maintain contact with gutta-percha and extended to the outside as one of two working electrodes. A stainless steel wire with the same dimensions of those of the copper wire, used as the other working electrode, was immersed into the background electrolyte from the center of the bottle. The electrical current between standard and experimental electrodes in canals was measured over a period of 10 days applying a conductivity meter. The Kruskal-Wallis test (p=0.05) determined whether there was a significant difference in microleakage among the groups and the Mann-Whitney U test (p=0.01) was used for multiple comparison grouping variables. Results: The results suggest that only the differences between the root canal filling techniques were statistically significant (p<0.01). There were no statistically significant difference in the sealers and the times of filling removal for post space preparation (p>0.01). Conclusion: The quality of the root canal filling is important for the integrity of the apical seal.