Although many bacteria are ureolytic, and in some cases urease acts as a virulence factor, the urease phenotype has not been analyzed in the anaerobic pathogen Clostridium perfringens. In this study, approximately 2% of C. perfringens strains, representing the principal biotypes, were found to harbor the urease structural genes, ureABC, and these were localized on large plasmids that often encode, in addition, the lethal epsilon or iota toxins or the enterotoxin. This represents the first report of a plasmid-encoded urease in a gram-positive bacterium. The C. perfringens enzyme was highly similar to the ureases of other bacteria and cross-reacted with antibodies raised against the urease purified from Helicobacter pylori. Urease production was inhibited by urea and induced under growth conditions where the availability of nitrogen sources was limiting. To date, this form of regulation has been observed only for chromosomal ureABC genes.