Objective: The aim of this study was to explore the effect of repeated sterilization on the surface and microstructural characteristics of two rotary nickel-titanium endodontic instruments. Material and methods: A total of 42 instruments of equivalent size corresponding to file size ISO 25 of ProTaper and K3 rotary system were investigated in this study. Twenty one files of each system( size F2 ProTaper and #25 K3) were divided as follows:Three files “as received” served as control.The remaining 18 files were equally divided into 2 groups; 9 files each, according to whether they were used to prepare root canal or not into group A and B. Each group was examined by Atomic force microscopy (AFM) and Scanning Electron microscope (SEM) to asses the effect of sterilization on the surface, On the other hand crystallographic and microstructural changes which are essential for there clinical performance were studied using Differential Scanning Calorimetry (DSC) and X-ray Diffraction (XRD). Results: The results obtained showed that repeated sterilization cycles had no significant effect on the phase and crystallographic changes of ProTaper and K3 files. On the other hand sterilization by autoclave had slight but no significant effect even on the surface topography of ProTaper and K3 files. Conclusions: Repeated sterilization cycles up to 3 cycles had no significant effect on the phase and crystallographic changes of ProTaper and K3 files as both NiTi files remains in Austenite form or a mixture of Austenite and R-phase at room temperature to 150oC which is above the sterilization temperature used in the study; thus no change in the main properties of the NiTi files regarding super-elasticity and shape memory which austenite and R-phases are responsible for in absence of the martensite phase. K3 files are rougher than ProTaper files and their surface deteriorate faster by use.