There are conflicting data on the occurrence of Enterococcus faecalis in the oral cavity of endodontic patients. This study investigated the prevalence of E. faecalis in multi-site oral samples (n = 136) from 41 endodontic patients using culture and polymerase chain reaction (PCR). Additionally, culturable strains were investigated for virulence traits. Overall, E. faecalis was detected in at least one tongue, oral rinse, or gingival sulcus sample in 68% of patients and in the root canals only in 5% of patients. In 21 patients from whom samples were obtained from all four sites, E. faecalis was detected in more tongue than gingival sulcus, oral rinse, and root canal samples (43, 14, 10, and 10%, respectively; p = 0.0148, chi(2)), and in proportionally greater numbers of patients with gingivitis/periodontitis compared to healthy periodontium (73% versus 20%; p = 0.03, Fisher's exact test). PCR was more sensitive than culture in detecting E. faecalis in oral samples (32% and 4%, respectively; p < 0.0001, McNemar's test). Multiple virulence traits were identified in culturable strains.