Fusobacterium nucleatum is a Gram-negative, non-spore-forming, nonmotile, obligatory anaerobic rod that is normally isolated from the oral cavity. Epidemiological studies have shown that this species is one of the most prevalent in primary root canal infections. The purpose of this study was to compare the effectiveness of bacteriological culture, 16S rDNA directed polymerase chain reaction and checkerboard DNA-DNA hybridization for detection of F. nucleatum strains in infected teeth associated with periradicular lesions. Thirteen single-root teeth from adult patients, all having carious lesions, necrotic pulps, and radiographic evidence of periradicular bone loss were included in this study. Combining all methods, the results indicated that F. nucleatum was present in approximately 31% (4 of 13) of the specimens. Incidence of F. nucleatum in root canal infections, as evaluated in this study by polymerase chain reaction, culture, and DNA-DNA hybridization, was 15.4%, 15.4%, and 10.0%, respectively. Our data demonstrated that no method used herein could be considered superior for detecting F. nucleatum directly from clinical samples. However, the small number of samples examined and the low prevalence that was observed should be considered.