Porphyromonas gingivalis (formerly Bacteroides gingivalis) degrades numerous protein substrates including collagen, fibrinogen, fibronectin, gelatin, casein, immunoglobulins and complement components. In order to clone one or more of these protease genes, a genomic library was constructed with Sau3A1 restriction fragments of chromosomal DNA from P. gingivalis ATCC 33277 ligated into the temperature-regulated vector pCQV2, and expressed in Escherichia coli DH5 alpha mcr. The electro-transformants (3 x 10(4)) were screened for general protease activity on Luria broth agar containing ampicillin (50 mg/l) and sodium caseinate (2%). One casein-hydrolyzing clone was detected and subcultured, and the activity of the cell extracts was characterized. We were able to show that the protease-positive clone, (pTEM1), had broad substrate specificity. Colorimetric assays indicated the hydrolysis of azocoll, azocasein, collagen, elastin-congo red and artificial substrates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to confirm that collagen, casein, fibrinogen and fibronectin were degraded by the clone.