We previously reported that the in vitro antimicrobial activity of a 2.0% chlorhexidine endodontic irrigant was equivalent to that of 5.25% sodium hypochlorite. The purpose of this study was to determine if chlorhexidine irrigants could instill substantive antimicrobial activity in instrumented root canals in vitro. Human teeth were instrumented using 2.0% or 0.12% chlorhexidine as irrigants. After instrumentation, the root canals were filled with sterile water, and samples of the root canal fluid were absorbed with paper points at 6, 12, 24, 48, and 72 h after treatment. The paper points were assayed for antimicrobial activity by placing them on agar plate surfaces inoculated with Streptococcus mutans and measuring zones of inhibition. Antimicrobial activity was present in all 2.0% chlorhexidine-treated teeth throughout the 72-h testing period and in most teeth, in relatively lower concentrations, for 6 to 24 h after irrigation with 0.12% chlorhexidine. These results indicate that chlorhexidine instills substantive antimicrobial activity when used as an endodontic irrigant.