Objective: This study aimed to compare the efficacy of Manual technique (Gates-Glidden and Hedstrom files), Protaper universal rotary retreatment system and R-Endo retreatment system in removing gutta-percha with different canals sealer, zinc oxide eugenol and AH Plus sealer. Materials and Method: Ninety-six extracted human roots were instrumented by ProTaper rotary files to (F3) and the samples were randomly divided into six groups of 16 roots for each groups A, B and C: gutta-percha and AH plus sealer, groups D, E and F: gutta-percha and Endoseal. Removal of gutta-percha was performed the following techniques; (group A and D) ProTaper retreatment instruments, (group B and E) R-Endo instruments and (group C and F) Gates-Glidden with Hedstrom files. The roots were split longitudinally. The area of remaining filling was evaluated by using stereomicroscope at three levels in the canal and time of retreatment was determined in each group. Results: No system completely removed the root filling material from root canal walls. No significant differences were observed between the rotary systems in terms of the area of filling material left within the canals (P > 0.05). In case of middle third of the canal, groups reinstrumented by manual technique (C and F) showed significant when compared to groups (reinstrumented with protaper retreatment files (A and D) Conclusion: No system removed the root filling materials entirely. ProTaper files and R-Endo were more efficient and faster than Gates-Glidden drills with H-files in root canal filling removal. Epoxy resin based sealer, was generally more difficult to remove than zinc-oxide and eugenol based sealer.