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Reports on the prevalence of Enterococcus faecalis in root canals vary considerably, potentially because of variations in clinical sampling and sample analysis methods. This study compared culture and real-time quantitative polymerase chain reaction (qPCR) to detect and quantify E. faecalis in the same root canal sample. Consecutive root canal samples obtained from primary infection (n = 40) and retreatment (n = 48) cases were divided into two equal aliquots that were independently analyzed using culture and qPCR by investigators blinded to the analysis results of the other sample. E. faecalis was detected in 10.2% and 79.5% of samples by culture and qPCR, respectively (p < 0.0001; McNemar's test). E. faecalis was detected in more retreatment than primary infection samples (89.6% versus 67.5%; p = 0.01, Fisher's exact test). qPCR reported a significantly higher prevalence of E. faecalis in endodontic samples than culture techniques.