This in vitro study in extracted human teeth was designed to compare the degree of dye penetration of root-end cavities prepared by CO2 laser, ultrasonic retrotip, or rotary instrument. Eighty-two human anterior teeth were prepared and obturated with laterally condensed gutta-percha and sealer. Root-end resection was performed at 90 degrees, 3 mm from apex. Root-end cavities were prepared by CO2 laser beam, ultrasonic retrotips, or bur and were filled with amalgam. The teeth were isolated and immersed in basic fuchsin at 36degreesC for 48 hours. The roots were split longitudinally and examined by a stereomicroscope. Leakage was quantified by linear measurement of dye penetration with the aid of image-analyzing software. Apical leakage in the CO2 laser group was significantly less than in the ultrasonic retrotip and bur groups. The difference between the ultrasonic and bur groups was insignificant. Based on the results of this in vitro study, application of CO2 laser for root-end cavity preparation appears to decrease the apical leakage.