Yeast cultures of Histoplasma capsulatum var. duboisii and H. capsulatum var. capsulatum in collagen containing defined, semi-defined and complex media produced extracellular collagenolytic proteinases, assayed using 4-phenylazo-benzyloxycarbonyl-L-propyl-L-leucyl- glycyl-L-propyl-D-arginine, a specific collagenase substrate. Significant levels of hydroxyproline were measured in the cultures and clear zones of hydrolysis were produced in collagen buffer agar by the crude enzyme preparations. Hydrolysis of casein and bovine serum albumin at pH 8 suggests the presence, in the crude enzymes, of multiple proteinases rather than a collagenase with broad substrate specificity since collagenolytic activity was not detected at pH 5 and above. Collagenolytic activities in the crude enzymes of both fungi were optimal at pH 4, 40 degrees C and were inhibited by EDTA, phosphoramidion and aprotinin indicating a metallo-serine nature. The molecular weights, estimated by column chromatography, were both 17 kD. The enzymes probably constitute a shared antigen. A probable role in the pathogenesis of histoplasmosis is discussed.