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The purpose of this study was to compare the incidence and mode of instrument separation of the two nickel-titanium (NiTi) rotary systems that were used according to a predefined schedule of clinical use by the same group of operators. A total of 166 ProFile and 325 ProTaper instruments, discarded from this endodontic clinic over 17 months, was analyzed. The incidences of instrument separation were 7% for ProFile and 14% for ProTaper (p < 0.05). The proportion of unwinding defects was 5% in ProFile and 0.3% in ProTaper instruments (p < 0.01). Flexural fatigue was implicated in the majority of separations in both groups. The results of this study indicated that while ProTaper was more likely to separate without warning, ProFile tended to exhibit unwinding of flutes more frequently.