The aim of this study was to evaluate the effect of root canal obturation with or without prior calcium hydroxide (Ca(OH)(2)) or 2% chlorhexidine (CHX) on the persistence of bacteria or its DNA in infected dentinal tubules. Canals of 85 extracted teeth were instrumented and inoculated with 10(4) cells/mL of Enterococcus faecalis. Teeth were incubated at 37 degrees C for 21 days and divided into 3 groups of 25 teeth plus controls. Teeth in group 1 were obturated immediately with gutta-percha (GP) and AH-Plus (Maillefer, Dentsply, Tulsa, OK). In group 2, Ca(OH)(2) was placed for 7 days before obturation. In group 3, 10 minutes of irrigation was performed with CHX performed before obturation. After incubation, GP was removed, and dentin specimens were collected and analyzed with culturing and polymerase chain reaction (PCR). No growth occurred in any cultures. By using PCR, E faecalis was detected in fewer roots in group 3 than in groups 1 or 2 (chi(2), p = 0.05); 2% CHX treatment followed by obturation was more effective in removing E faecalis DNA than placement of Ca(OH)(2) or immediate obturation.