INTRODUCTION: The aim of this study was to evaluate the amount of dentine removed after canal preparation using stainless steel (SS) hand instruments or rotary ProFile instruments. MATERIALS AND METHODS: Thirty-six extracted human teeth with root canal curvatures less than 30º were embedded in clear polyester resin. The roots were cut horizontally at apical 2, 4 and 7 mm. Dentin thickness was measured at each section and the sections were accurately reassembled using a muffle. Root canals were randomly prepared by SS hand instruments or rotary ProFile instruments. Root sections were again separated, and the remaining dentin thickness was measured. Mann-Whitney U and t tests were performed for analytic comparison of the results. RESULTS: The thickness of removed dentin was significantly different between the two used methods (P<0.05). Significantly greater amounts of dentin was removed mesially in all sections in hand instrumentation group (P<0.001). CONCLUSION: ProFile rotary instrumentation prepares root canals with a greater conservation of tooth structure.