The purpose of this study was to determine if the Nd:YAG laser was capable of disinfecting contaminated root canals in vitro. Eighty canals of extracted single-rooted teeth were prepared to size 35 K-file. The teeth were sterilized with ethylene oxide gas and then inoculated with Bacillus stearothermophilus. Eight groups were treated as follows: 1, sterility control; 2, positive control, no treatment; 3, hand instrumentation with sterile water filling chamber; 4, hand instrumentation with NaOCl (5.25%) filling chamber; 5, ultrasonic instrumentation with sterile water; 6, ultrasonic instrumentation with NaOCl; 7, laser exposure with sterile water; and 8, laser exposure with NaOCI. All instrumentation times were 1 min. Teeth were then flushed with 1.0 ml of sterile water onto blood agar plates, and the number of colony forming units were determined. Results indicate that: (i) all groups treated with NaOCI effectively inhibited the growth of B. stearothermophilus; (ii) hand and ultrasonic instrumentation with sterile water did not effectively disinfect root canals; and (iii) the Nd:YAG laser was effective in inhibiting the growth of B. stearothermophilus. The effects of the Nd:YAG laser on other bacteria more commonly found in root canal systems should be evaluated.