An extracellular proteinase from a Xanthomonas maltophilia strain isolated from soil was purified by ammonium sulfate precipitation, gel exclusion, and ion exchange gel chromatography. The enzyme appeared homogeneous upon polyacrylamide-gel electrophoresis. Its molecular weight was estimated to be 36,000 by the gel filtration assay and 40,000 by polyacrylamide-gel electrophoresis after denaturation: it was a monomeric protein. Highly active at alkaline pH with casein as the substrate, it was inactivated by serine-site inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. Like serine proteases, it hydrolyzed esters such as the beta-naphthyl acetate. Moreover, like metalloproteases, the enzyme was rapidly inactivated by ethylene diamine tetraacetate, and the activity was partially restored by calcium. These original characteristics have not often been reported among other gram-negative bacteria.