Using a colorimetric substrate azocoll, we assessed the proteolytic activities of culture filtrates from 12 staining fungi grown in liquid media supplemented with protein. All the fungi produced some extracellular proteolytic activity, but the level of activity varied among the different species. When the activities were expressed unit of fungal dry weight, the maxima for proteinase activities were recorded during the active growth phase of the fungi. Proteinase samples prepared from nine fungal strains by ammonium sulfate precipitation were examined by native PAGE and IEF electrophoresis followed by x-ray film overlays. At least two or three proteolytic bands were detected in each sample. Temperature tests showed maximum activities around 37-40 degrees C. Proteinase-inhibition studies suggested that the majority of the enzymes were metal-activated serine-type proteinases.