Actinobacillus actinomycetemcomitans (Aa) has been implicated in most cases of localized juvenile periodontitis and some cases of severe adult periodontitis and refractory periodontitis. The Aa leukotoxin plays an important role in the pathogenesis of Aa associated periodontal disease. Rapid detection of Aa in a periodontal pocket is hampered by the slow growth and fastidious nature of this bacterium. In this study, we developed a rapid, sensitive, nonradioactive polymerase chain reaction (PCR) to identify a unique As sequence directly from subgingival plaque samples. Two oligonucleotide primers derived from DNA sequences of the leukotoxin gene were used in the PCR. The Aa-specific DNA fragments were analyzed by agarose gel electrophoresis and then visualized under 302 nm ultraviolet light after staining with ethidium bromide. In the 12 subgingival plaque samples screened, the Aa-specific sequences were found in five out of nine sites with periodontitis. No Aa-specific sequence was found in three healthy sites. The specificity of the amplified DNA fragments was confirmed by direct DNA sequencing. These results indicated that the PCR technique should assist in the rapid detection of Aa in subgingival plaque samples. Moreover, combined with direct DNA sequencing, this method can be used to study the molecular epidemiology of this periodontal pathogen.