The purpose of this study was to investigate the influence of cervical preflaring on determination of the initial apical file in maxillary lateral incisors. Forty human lateral incisors with complete root formation were used. After standard access cavities, a size 06 K-file was inserted into each canal until the apical foramen was reached. The WL (WL) was set 1 mm short of the apical foramen. Four groups (n=10) were formed at random, according to the type of cervical preflaring performed. Group 1 received the initial apical instrument without previous preflaring of the cervical and middle root canal thirds. Group 2 had the cervical and middle root canal thirds enlarged with nickel-titanium Orifice Opener instruments. Group 3 had the cervical and middle root canal thirds enlarged with Gates-Glidden drills. Titanium-nitrite treated, stainless steel LA Axxess burs were used for preflaring the cervical and middle root canal thirds of group 4. Each canal was sized using manual K-files, starting with size 08 files with passive movements until the WL was reached. File sizes were increased until a binding sensation was felt at the WL, and the instrument size was recorded for each tooth. The apical region was then observed under a stereoscopic magnifier, images were recorded digitally and the differences between root canal and maximum file diameters were recorded (in mm) for each sample. Significant differences were found between the groups regarding the anatomical diameter at the WL and the first file to bind the canal (p = 0.01). The major discrepancy was found when no preflaring was performed (0.1882 mm average). Canals preflared with Orifice Opener instruments (0.0485 mm average) and Gates-Glidden drills (0.1074 mm average) also showed great discrepancy. The LA Axxess burs produced the smallest differences between anatomical diameter and first file to bind (0.0119 mm average). Instrument binding technique for determining anatomical diameter at WL was not accurate. Preflaring of the cervical and middle thirds of the root canal improved anatomical diameter determination; the instrument used for preflaring played a major role on determination of the anatomical diameter at the WL. Canals preflared with LA Axxess burs created a more accurate relationship between file size and anatomical diameter.