This study was conducted to determine the bacterial reduction using Profile GT® files and a strict irrigation protocol utilizing 5.25% NaOCl and EDTA. The additive antibacterial effect of Ca(OH) 2 was also evaluated. In addition, the study compared the bacterial reduction with the GT protocol versus larger instrumentation. Thirty-one subjects with apical periodontitis were recruited. Bacterial samples were taken upon access (S1), after instrumentation and a strict irrigation protocol (S2), and following Ͼ1 wk of Ca(OH) 2 (SC). A log 10 transformation of colony forming units was done since sample bacterial counts are not normally distributed. At S1, 93.55% of canals sampled bacteria. At S2, 52.72% of the cases sampled bacteria. At SC, 14% of the cases cultured bacteria. The McNemar test showed a significant reduction (p Ͻ 0.0009) in bacteria between S1 and S2. This was also true between S2 and SC (p Ͻ 0.0019). It was concluded the GT protocol significantly reduced the number of bacteria in the canal but failed to render the canal bacteria free in more than half of the cases Ca(OH) 2 application significantly further reduced bacteria. Lastly, large apical instrumentation removed more bacteria than small apical instrumentation.This study was funded in part by a grant from The Amer- (1-4).Because bacteria are the cause of apical periodontitis, it is logical that the elimination of bacteria would cure apical periodontitis. A tooth without periapical radiolucency has a higher endodontic success rate upon treatment than a tooth with a radiolucency (5-8). Likewise, teeth that are obturated after a negative culture have a better prognosis than those obturated following a positive culture (9 -11). Although there is substantial evidence that a negative culture does not equate to bacteria free (12-14), there is a threshold level of bacteria below that gives a negative sample and a success rate similar to teeth treated without apical periodontitis (11).The current technique for bacterial reduction includes instrumentation, irrigation with NaOCl and an intracanal antimicrobial medication. showed that each step leads to less bacteria and eventually a canal that does not sample bacteria. In a 5 yr follow-up study, root canal samples that gave a negative culture following these steps were recalled to evaluate healing. Ninety-five percent of these cases showed complete radiographic healing or a decrease in the radiolucency size (10).A recent series of studies have been carried out where NiTi instruments were used in place of stainless steel files (19 -21). Similar to the Bystrom and Sunqvist (15) findings, Dalton et al. (19) found that while instrumentation decreased the number of canals that cultured bacteria, instrumentation alone does not predictable result in canals that did not culture bacteria. Shuping at al (20). evaluated the bacterial reduction following the addition of 1.25% NaOCl irrigation and Ca(OH)2 to NiTi instrumentation. Similar to previous studies, Shuping et al. (20) found that instrumentation and i...