The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.