Forty-five Streptomyces strains, including representatives of the plant pathogens S. acidiscabies, S. scabies, and S. ipomoea, were screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin. Initially, all strains were tested for production of extracellular esterase in liquid shake (250 rpm) cultures at room temperature in defined (glycerol-asparagine) or complex (tryptone-yeast extract with or without addition of mannitol) broth media supplemented with either tomato or apple cutin. Esterase activity was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. Of the five strains exhibiting highest esterase activity, four (S. acidiscabies ATCC 49003, S. "scabies" ATCC 15485 and IMRU 3018, and S. badius ATCC 19888) were confirmed to produce enzymes with cutin-degrading activity (cutinases). Confirmation of extracellular cutinase production was accomplished by use of a new high-performance liquid chromatography method for separation and quantification of released cutin monomers. Monomer identification was confirmed by GC/MS analyses. Cutinase production was induced 2- to 17-fold by inclusion of cutin in the media. To our knowledge this constitutes the first report of cutinase production by Streptomyces spp. other than S. scabies.