Statement of Problems: The extracted human teeth are commonly used in endodontic research and preclinical education. The Center for Disease Control (CDC) recommends sterilizing the extracted teeth by storing in 10% formalin for 2 weeks or autoclaving.Purpose: The purpose of this study was to determine the potential effect of storage in formalin or autoclaving of the extracted human teeth on the apical seal integrity of the obturated canals.Materials and Method: A total of 83 extracted single–rooted teeth, free of visible fracture, caries or large restoration or root canal treatment were collected, and temporarily stored in normal saline for no longer than 48 hours before categorizing them in experimental and control groups. The teeth were divided into three experimental groups of 25 each, and placed in 10% formalin for 2 weeks or autoclaved or immediately prepared. Eight teeth served as positive and negative controls. Preparations for all the teeth consisted of decornation, canal instrumentation by protaper system and obturation with lateral compaction technique. The teeth were immersed in India ink and cleared. Then the greatest extent of linear dye leakage was measured. Leakage measurements were evaluated for significant differences using ANOVA with Tukey's HSD Post Hoc analysis.Results: There was less dye leakage in the teeth stored in formalin as compared to autoclaved and freshly extracted teeth. No statistical significant differences in apical microleakage between the three experimental groups were observed ( p > 0.05).Conclusion: The results of this study revealed that storage of the extracted teeth for research and educational purpose in formalin or autoclaving had no effect on apical microleakage of the obturated teeth.Key words: Apical leakage, Autoclave sterilization, Formalin