Background: Matrix metalloproteinases (MMPs) participate in extracellular matrix degradation in physiological and pathological conditions. The available evidence suggests that MMP‐13 plays a significant role in both the initiation and progress of bone resorption. The aim of our study was to identify the presence of MMP‐13 in adult patients with untreated chronic periodontitis. We also determined the activity of MMP‐13 present in lesions undergoing episodic attachment loss in gingival crevicular fluid (GCF) samples.Methods: After monitoring at 2 and 4 months, 21 patients showed destructive periodontitis (periodontally affected sites presenting at least two sites with ≥2 mm clinical attachment loss), and GCF samples were collected both from active and inactive sites (21 GCF samples, each). GCF was collected during a 30‐second interval using a paper strip, and an immunofluorescence assay was performed to determine the basal activity of MMP‐13 and the relationship between 4‐aminophenylmercuric acetate (APMA)‐activated total MMP‐13 and basal MMP‐13 activity. Gingival tissues from five patients were fixed in formalin and MMP‐13 expression was demonstrated using immunohistochemistry and in situ hybridization. MMP‐13 molecular forms were examined by Western immunoblotting with monoclonal antibodies.Results: MMP‐13 was found in 100% of GCF samples from patients with chronic periodontitis. Active sites, associated with tissue destruction, had significantly higher proportions of active MMP‐13 and MMP‐13 activity levels than their inactive counterparts (1.49 versus 1.17 ng fluorescent product, respectively; P <0.05). Western blot, immunohistochemical staining, and in situ hybridization confirmed the presence of MMP‐13 in periodontal disease, with observable differences between periodontitis and healthy subjects. MMP‐13 immunoreactivities were seen mainly as 55 and 48 kDa, corresponding to partially and fully activated forms, respectively, and a smaller proportion of 60‐kDa proenzyme form.Conclusion: MMP‐13 activity in GCF samples was significantly increased in active sites from progressive periodontal disease, supporting its role in the alveolar bone loss developed in this disease.