The purpose of this study was to investigate the mode of failure of three brands of nickel-titanium (NiTi) instruments (ProTaper [Dentsply Maillefer, Ballaigues, Switzerland], ProTaper for Hand Use [Dentsply Maillefer], and K3 [SybronEndo, Orange, CA]) that separated during clinical use. A total of 79 fractured instruments were collected from three endodontic clinics over 16 months. The fracture surface of each fragment was examined by scanning electron microscope. Most of the rotary instruments (78% of K3 and 66% of ProTaper) failed because of fatigue fracture, whereas 91% of NiTi hand instruments failed as a result of shear (p < 0.01). The fracture mode of shaping files in rotary ProTaper was different between two different clinics (p < 0.05). All surfaces with fatigue fracture (n = 47) revealed the presence of either one or two crack origins. The vast majority (86%) of K3 fatigue failure had two crack origins that could be found not only at the cutting edge but also at various places along the flute compared with only 28% of ProTaper showing multiple crack origins (p < 0.01). The latter showed one crack origin in 81% of the fatigued shaping files but only 37% for finishing files (p < 0.01). The results of this study indicated that the failure mode of NiTi instruments is related to preparation technique.