Detection of putative pathogens is critical for delineating the etiology and progression of periodontitis. In the present study, we have used a polymerase chain reaction (PCR) assay utilizing primers specific for the lkt A gene of Actinobacillus actinomycetemcomitans, the fimbrial gene of Porphyromonas gingivalis, and tdp A gene of Treponema denticola in order to determine the presence of these pathogens in subgingival plaque samples from periodontitis sites. These gene specific primers were also used to assess the detection of different strains of bacteria in the PCR assays. Primers for P. gingivalis detected P. gingivalis strain 33277, but no product was detected when primers were used with extracts from 4 species of Capnocytophaga, 3 species of Prevotella, 2 species of Porphyromonas other than P. gingivalis, Bacteroides levii, Escherichia coli, 3 strains of A. actinomycetemcomitans and 3 strains of T. denticola. PCR analysis using primers for the lkt A gene of A. actinomycetemcomitans also did not result in a product with any of these bacteria with the exception of a positive result with 3 different strains of A. actinomycetemcomitans. Primers selected from the tdp A gene of T. denticola did not identify any of the bacteria strains tested except T. denticola serovars a, b, and c. Thus, these primers were shown to amplify gene segments that are specific to either P. gingivalis (33277), A. actinomycetemcomitans (33384, 43717 and 43718) or T. denticola (35405, 33521 and 35404). The PCR assay may be used to rapidly detect the presence of periodontal pathogens in the future.