As a diagnostic test, "Periocheck" can detect the N-carbobenzoxyglycyl-glycy-arginyl peptidase that is produced by Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus. The aim of this study was to clarify the relationship between peptidase activity and attachment loss. After Phase 1 and surgical therapy, a total of 111 sites from 47 adult periodontitis patients were divided into four groups according to peptidase activity (trypsin unit, TU): A, < 0.1 TU; B, 0.1-0.2 TU; C and D > or = 0.2 TU. All sites in groups A, B, and C were untreated, whereas both subgingival 3% hydrogen peroxide irrigation and 2% minocycline application were undertaken every 45 days throughout the experiment in group D. All subjects were recalled at 3-month intervals. Peptidase activity and clinical assessments were measured for the 18-month period. Significant attachment loss associated with high values of the peptidase activity was found through the experimental period in groups B and C. In contrast, no obvious change of attachment loss was found in groups A and D following low peptidase activity at 6, 12, and 18 months. The mean attachment loss throughout the 18-month period was 0.22 mm in group A, 1.04 mm in group B, 1.53 mm in group C, and -0.35 mm in group D. Probing depth and percentages of bleeding on probing significantly increased in group C, whereas they decreased in group D. This peptidase test displayed a 77.8% sensitivity and 68.6% specificity regarding the detection of > or = 1 mm attachment loss with a cut-off value of 0.1 TU. Multiple linear regression analysis showed a close relationship between peptidase activity and predictable attachment loss within a 12-month period. These findings suggest that this peptidase test is useful in identifying the risk sites for predictable attachment loss.