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This in vitro investigation assessed the efficacy of removing radioactively labeled bacteria from infected canals with 2 engine-driven rotary nickel titanium instrumentation techniques differing in sequence and apical enlargement size.A standard quantity of 3H-thymidine-labeled Enterococcus faecalis (3.70 × 104 cpm, 2.0 × 107 colony-forming units) was used to inoculate the mesiobuccal canals of 50 extracted mandibular molars. The teeth were incubated for 5 days to allow infection of the surrounding dentin from the canals. Five of the teeth were used as controls to determine the number of cycles of irrigation and drying necessary to reduce the 3H counts recovered from the canals to baseline levels. After this process, the unbound bacteria in the root canals of the remaining 45 teeth then were washed out with buffer until baseline levels of radioactivity were obtained. The mesiobuccal root of 1 of these 45 teeth was removed, decalcified, and digested, and the total radioactivity released from the root dentin was measured. Of the remaining 44 teeth, 22 then were instrumented with GT and Profile (Dentsply/Tulsa Dental Co, Tulsa, Okla) instruments to apical size #35 (group 1) and 22 teeth with Pow-R instruments (Moyco/Union Broach, York, Pa) to apical size #50 (group 2), in the presence of a standard quantity of phosphate-buffered saline solution placed in the canal. After instrumentation, the medium from each canal was collected with paper points and its radioactivity was counted with liquid scintillation spectrometry.The mean 3H level recovered with instrumentation of canals in group 1 was 75 cpm (± 29, standard deviation) and in group 2 was 123 cpm (± 50, standard deviation). A 2-tailed Mann-Whitney test indicated that the radioactivity of samples from group 2 was significantly higher than that of samples from group 1.The results suggested that instrumentation to an apical size of #50, as performed with the Pow-R instruments, was more effective in debriding infected root canals than instrumentation to an apical size of #35, as performed with the GT and Profile instruments.