Nora M. Abd-Allah¹, Abeer A. El-Fallal², Shaymaa E. Elsaka^{3 1}Dentist at Mansoura General Hospital, B.D.S. , Faculty of Dentistry, Mansoura University. ²Professor of Dental Biomaterials, Faculty of Dentistry, Mansoura University. ³ Associate Professor of Dental Biomaterials, Faculty of Dentistry, Mansoura University. Abstract: Objectives:To evaluate the effect of autoclaving, gamma radiation, and ultraviolet sterilization methods on surface properties of traditional and new twisted NiTi rotary endodontic instruments. Methods:A total of 64 NiTi rotary endodontic instruments, 32 Revo-S (RS, Micro-Mega, Besancon, France) and 32 Twisted Files (TF, SybronEndo, Orange, CA, USA) of equivalent size corresponding to file size 25, and taper 0.06 were analyzed. Instruments were evaluated in as-received condition and after one cycle of autoclaving, gamma radiation, and ultraviolet sterilization methods. After sterilization, the specimens were subjected to scanning electron microscope (SEM) , atomic force microscopy (AFM), and roughness average (Ra) value of the scanned surface profiles were recorded. Data were analyzed by means of ANOVA, least significant difference (LSD), and T-test. Results:SEM observations revealed the presence of surface irregularities in all instruments. After different sterilization methods, SEM examination revealed that there were no obvious changes observed in surface morphology as a function of sterilization method. Ra values of both RS and TF in all sterilized experimental groups were significantly different compared with Ra values of control group (P = 0.0001). There were statistically significant difference between autoclaving group and other two groups (gamma radiation and ultraviolet radiation) where P = 0.0001. However, there was no significance difference between gamma radiation and ultraviolet radiation groups (P > 0.05). Conclusions:Autoclave sterilization method affected surface properties of both traditional and new twisted NiTi rotary endodontic files than gamma radiation and ultraviolet sterilization methods. Keywords:NiTi, RS, TF, sterilization, SEM, AFM.