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Established human cell linesderived from a transitional cell carcinoma (J82), a squamous carcinoma (SCaBER), and a normal urothelium (HCV‐29) were used to assess the relative cytotoxicity and tissue specificity of putative virulence determinants of P. gingivalisW83. Intact cells of W83 had no effect on any of the cell lines, whereas disrupted cells caused extensive cytotoxicity particularly to monolayers of HCV‐29 and J82. The purified cysteine proteinase, gingivain, caused marked disruption of the basement membrane of the SCaBER monolayers but had no cytotoxic effects. Use of the thiol‐inhibitor, 2,2′‐dipyridyl disulphide revealed that the effects observed with the vesicles and the culture supernatant were due to the presence of the cysteine proteinase. The attachment of vesicles to the SCaBER cells was evident in electron micrographs. Shortchain volatile fatty acids added in concentrations equivalent to those present in the culture supernatant had no effect on any of the cell lines tested. Culture supernatants obtained from high speed centrifugation (150,000 × g) showed no cytotoxic effects. This was in marked contrast to the supernatant obtained by lower sedimentation (18,000 × g), which damaged all monolayers tested. These results suggest that these cell lines are potentially useful for assessing putative virulence determinants of P. gingivalis and other periodontal pathogens. J Periodontol 1992;63:44–51.