Aim To assess the presence of Enterococcus faecalis after root canal treatment in single or multiple visits in an ex vivo model. Methodology Forty-five premolar teeth were infected ex vivo with E. faecalis for 60 days. The canals were then prepared using a crowndown technique with System GT and Gates-Glidden burs and irrigated with 2% chlorhexidine gel. The specimens were divided into five groups (G1, G2, G3, G4 and G5) according to the time elapsed between chemical-mechanical preparation and root canal filling, the irrigant solution used and the use or nonuse of a calcium hydroxide intra-canal medicament. The teeth were then root-filled and incubated for 60 days at 37 degrees C. Dentine chips were removed from the canal walls with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing Brain-Heart Infusion broth. These samples were placed onto agar plates and colony forming units were counted after 24 h at 37 degrees C. Data were ranked and analysed using the Kruskal-Wallis statistical test. Results Enterococcus faecalis was recovered from 20% (three of 15 specimens) of G1 (chlorhexidine irrigation and immediate root filling in a single visit), 25% (four of 15 specimens) of G2 (chlorhexidine irrigation and filling after 14 days use of a calcium hydroxide dressing in multiple visits), 40% (two of five specimens) of G3 (chlorhexidine irrigation and filling after 7 days), 60% (three of five specimens) of G4 (saline irrigation and filling after 7 days) and from 100% (five of five specimens) of G5 (saline irrigation and immediate filling without sealer). Conclusions Neither single- nor multiple-visit root canal treatment ex vivo, eliminated E. faecalis completely from dentinal tubules. Up to 60 days after root filling, E. faecalis remained viable inside dentinal tubules. When no sealer was used, E. faecalis presented a higher growth rate.3810697704