Treponema denticola and Bacteroides gingivalis are among the few recognized species found in periodontal pockets that can hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). We determined the presence of these periodontal pathogens in BANA-positive and -negative plaque samples through the use of indirect immunofluorescent antibody techniques. Eighteen of 27 diseased sites gave BANA-positive reactions, and 9 gave BANA-negative reactions. T. denticola was present in 16 of 18 BANA-positive reactions, whereas B. gingivalis was detected in 9 of the 18 BANA-positive reactions. T. denticola was present in 1 and B. gingivalis in 2 of the 9 BANA-negative reactions. Neither organism was detected in the 19 healthy sites that were negative for BANA. All measured differences between BANA-positive and BANA-negative plaques obtained in the same individuals were statistically significant. The accuracy of the BANA test, compared with clinical parameters such as bleeding upon probing and increased probing depth, was about 80%. The accuracy of the test in detecting the presence of T. denticola was 93%, for B. gingivalis, 76% and for T. denticola and/or B. gingivalis, 96%. This study indicated that BANA-positive plaques were associated with the presence of T. denticola and/or B. gingivalis, that T. denticola was found at a greater frequency and levels in BANA-positive plaques than B. gingivalis, and that the presence of these organisms was associated with clinical disease.