Objectives: The purpose of this paper is to evaluate in vitro the influence of various substances used in root canal preparation on the cut efficiency of manual endodontic files. Methods: This was achieved through the analysis of loss of mass and time of preparation in human teeth. Forty (40) canine teeth were divided into four groups according to chemical substance used as following: G1 (NaOCl 1%); G2 (RC-Prep); G3 (Endo-PTC in Carbowax base) and G4 (Endo-PTC in Carbopol base). The specimens were weighted both before and after preparation, thus making possible to obtain the mass difference which was divided by the preparation time for each specimen (in seconds), hence determining the cut efficiency (mg/s). The results were subjected to ANOVA analysis, followed by Tukey Test (a= 5%). Results: The Endo PTC in Carbopol base group showed the best cut efficiency (2.64 mg/s). A statistically meaningful difference was found between group G4 and the others (p< 0.05). Statistical differences amongst groups G1 (1.75 mg/s), G2 (1.74 mg/s) and G3 (1.82 mg/s) were not found for p>0.05. Conclusion: The cut efficiency of manual endodontic instruments is affected by the chemical substance used and the substitution of Carbowax for Carbopol made Endo PTC cream more efficient as an auxiliary on the root canal shaping. Supporting Agency: Fapesp (03/04433-7).