Sporothrix schenckii, mainly in the yeast form of the organism, produced extracelluilar proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being .indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pl at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins. proteinases, 200-ml samples of BSA-containing media in 500-ml flasks were incubated under the same conditions as described above for 1 week.Assay of proteinase activity. Proteinase activity was assayed with Azocoll (Sigma) as a substrate. A 5-mg sample of Azocoll in 50 mM citric acid-100 mM disodium phosphate buffer was incubated with 50 to 100 ,ul of enzyme solution (total volume, 1.0 ml) for 1 h at 37°C. Incubation was terminated by the addition of 2 ml of cold distilled water. After centrifugation at 1,500 x g for 10 min, released dye in the supernatant was measured at 520 nm with a Hitachi spectrophotometer (model 100-60). One unit of proteinase activity was defined as the amount of enzyme which produced an increase in absorbance of 0.10 under the abovementioned conditions. Determination of protein concentration. Protein concentration was determined by the method of Lowry et al. (9) (Amicon Corp., Lexington, Mass.). After centrifugation at 1,500 x g for 15 min, the supernatant was applied to a DEAE-Sepharose CL-6B column (1.2 by 11 cm) which had been previously equilibrated with the same 4104