Collagenolytic enzymes released by neutrophils are associated with the destruction of periodontium in periodontal diseases. Measurement of these enzymes in gingival crevicular fluid (GCF) could be used to test for periodontal diseases and thereby simplify diagnosis. To test this hypothesis, gelatinase (MMP-9) was analyzed in GCF samples with a simple assay system. GCF was collected by a mouthrinse method from 10 patients with gingivitis (G); 10 well-treated and maintained periodontitis patients (TP) without detectable loss of attachment; and 9 patients with recurrent loss of periodontal attachment (>2 mm) and/or abscess formation (RP). Clinical measurements including tooth mobility (MOB) and gingival attachment level (GAL) were made monthly for a maximum of 10 months. Active and latent forms of gelatinase were measured by a functional assay using gelatin substrate-gel enzymography and the activities were quantified by laser densitometry. Reproducibility analysis demonstrated that the assay (inter-gel, inter-assay, inter-scan) and diurnal variations were small compared to biological variation. The presence of active gelatinase was detected in 97.8% of TP samples, 86.4% of RP samples, but in only 11.4% of G samples. In addition, the mean active gelatinase activity was found to be significantly higher (p<0.001) in the RP (71 006 U) than the TP (43814 U) groups, both of which were higher (p< 0.001) than the G group (2824 U). During periods of attachment loss, samples from the RP group exhibited a 2-fold increase of mean active gelatinase activity (129414 U). Correlation and regression analyses demonstrated that active gelatinase activity was most strongly associated with loss of GAL (r = 0.52, p<0.0001) and to a lesser degree with mean MOB (r = 0.35, p<0.03). However, neither total (latent plus active) nor latent gelatinase levels were associated closely with any clinical parameters of periodontitis. Metronidazole treatment (250 mg, tid, for 7 days) of RP patients significantly reduced the level of active and latent gelatinase 4- to 6-fold (p< 0.002). These data indicate that measurement of active gelatinase in mouthrinse samples may provide a simple and robust diagnostic test for periodontal diseases and for assessment of treatment response.