Separation is a potential risk when using any instrument in the root canal system. Presently, there are no large clinical studies investigating the separation incidence of the ProTaper rotary file system or the number of times a ProTaper rotary file can be safely used. The purpose of this study was to determine if the number of uses affects the separation incidence of ProTaper rotary instruments. 4652 consecutively treated root canals were performed in an endodontic group practice over a 17-month period. Both the separation incidence and the number of uses were tracked for each file. The overall rate of instrument fracture in this study was 2.4% with no significant differences over the first four uses. The results of this study indicate that ProTaper rotary files may be safely reused at least four times. The size of the rotary file, among other factors, will determine how many times a particular file should be used.