This study conducted an in vitro analysis of the level of apical transportation in mesiobuccal roots of upper molars after manual instrumentation with stainless steel files, preparation with the K3 system, and with a reciprocating NSK handpiece. Sixty roots were mounted in muffles and then longitudinally sectioned. A digital image was acquired of the inner surface of one root half. After instrumentation, the inner surfaces of the same root halves were digitized with file sizes #30, #35, and #40. Each image obtained was superimposed over the corresponding preoperative image, and the apical transportation was measured. The Friedman and Wilcoxon tests were used to compare results for the file sizes, and comparison of the groups was made with the Kruskal-Wallis test, demonstrating statistically significant differences (P = .000). The stainless steel file sizes #35 and #40 caused significant apical transportation, and K3 system proved safe for apical preparation, with little deviation.