Treponema denticola, Porphyromonas (Bacteroides) gingivalis, and Bacteroides forsythus are among the anaerobic species frequently associated with adult forms of periodontal disease. These organisms hydrolyze the synthetic peptide benzoyl-DL-arginine-naphthylamide (BANA), and such enzyme activity can be detected in the plaque and related to clinical disease and the presence of spirochetes. In this investigation, the liquid BANA assay was compared with a commercially developed BANA assay which employed a paper format and which could be read after a 15-min incubation. In the paper format, strips of a Whatman filter paper were impregnated with BANA and strips of nitrocellulose paper were impregnated with fast black K salt. Both strips were applied lengthwise across a paper card (3 by 5 in. [7.6 by 12.7 cm]). The BANA strip at the bottom was inoculated with the test sample (pure culture, plaque), folded back so that it contacted the fast black strip, and then incubated for 15 min at 55 degrees C. T. denticola, P. gingivalis, and B. forsythus always gave a positive reaction, whereas 51 other plaque species were always negative. Six Bacteroides and Capnocytophaga species on occasion had weak reactions. The proportional agreement between BANA positiveness and clinical disease was similar for both the liquid and the paper assays. The sensitivity, specificity, and accuracy relative to the clinical standard of the liquid assay were 74, 76, and 77%, respectively, while those of the paper assay were 81, 78, and 80%, respectively. The paper assay was significantly associated with the presence of either T. denticola or P. gingivalis or both in the plaque samples, with a sensitivity of 85%, a specificity of 53%, and an accuracy of 79%. These findings indicate that a rapid paper assay for BANA hydrolysis gives data comparable to those obtained with the liquid BANA assay.