The purpose of this study was to analyze the incidence and mode of ProFile (Dentsply Tulsa Dental, Johnson City, TN) instrument separation during a predefined schedule of clinical use by the undergraduate students in a dental school over 4 years. A total of 3,706 ProFile instruments discarded from the same undergraduate students program between 2003 and 2007 were analyzed. The lateral and fracture surfaces of 12 separated instruments were examined by scanning electron microscopy, and the location of the fractures was recorded. The overall proportion of instrument defects was 1.3%; deformation without fracture occurred in 1% and separation in 0.3%. The majority of instrument defects occurred in size 20 (34/48). The ProFile instruments (10/12) failed mostly because of shear stress, whereas only two failed because of fatigue fracture. The results of this study indicated that NiTi rotary instrument system was successfully introduced into an undergraduate endodontic program. Small size files should be considered as single-use, disposable instruments because of the higher possibility of torsional deformation.