Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.