OBJECTIVE The purpose of this investigation was to evaluate the substantivity of chlorhexidine (CHX) within a root canal system and to assess how long the CHX remains antimicrobially effective. METHODS Bovine roots were sectioned and standardized to 8 mm. Sections, which served as controls, were treated with 1% sodium hypochlorite and 1 mol/L EDTA, then obturated with gutta percha and AH26 sealer. Experimental sections were treated similarly except they were placed in 2% CHX for 10 minutes prior to obturation. Control specimens were divided into 4 control groups and stored in saline for 1 day, 3 weeks, 6 weeks, and 12 weeks. Experimental specimens were divided into 4 groups and stored in saline for 1 day, 3 weeks, 6 weeks, and 12 weeks. After their respective storage periods, all specimens were halved and canal wall dentin was ground out with Peeso reamers. Dentin specimens were agitated in 700 microl of saline for 5 hours to release CHX. After centrifugation the supernatants were analyzed with UV spectrophotometry at 253 nm. To determine whether the CHX from dentin samples remained antimicrobial, the extracts from experimental and control groups were mixed with cultures of Enterococcus faecalis. RESULTS After 1 day of storage, the dentin extract contained approximately 0.0048% CHX. After 3, 6 and 12 weeks, dentin extracts contained approximately 0.0023%, 0.0016%, and 0.0010% CHX respectively. Extracts from the storage groups were found to be highly antimicrobial corresponding to the CHX concentration. CONCLUSIONS The results of this study indicate that CHX is retained in root canal dentin in antimicrobially effective amounts for up to 12 weeks.