The aim of this study was to evaluate the root canal anatomy of mandibular incisors before and after endodontic instrumentation, identifying regions inaccessible to the action of files (Critical instrumentation Area - CA) in a three-dimensional perspective. Thirty human mandibular central incisors were selected, assigned to two groups (n=15) and instrumented using ProTaper Universal rotary files. In the RX group, longitudinal digital radiographic images were obtained in the buccolingual (BL) and mesiodistal (MD) views. In the CT group, cross-sectional micro-computed tomography (µCT) images were obtained at 3, 9 and 15 mm from the apex. The canal area of the specimens was evaluated before and after instrumentation using digital images from each group. Data were analyzed using t-test, one-way ANOVA with subdivided parcels and Tukey's test (α=0.05). The canal area found in the MD radiographs was larger than in the BL radiographs, which was also confirmed in the transversal images (p<0.01). The CA was only detected in the MD radiographs and µCT scans. On the root canal configuration, a continuous reduction in the canal conicity was observed in BL radiographs, while in MD view there was a constriction at the cervical third and subsequent increase at the middle third (p<0.01). The conical shape of the root canal was observed only in the BL view. The canal enlargement in BL radiographs was not indicative of homogeneous instrumentation, since unprepared areas (CA) were also verified on the buccal and lingual walls in different images.