Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Species-specific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were Dialister invisus (81%), Synergistes oral clone BA121 (33%), and Olsenella uli (33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.