The basidiomycete Schizophyllum commune produces a variety of proteolytic enzymes. A number of these, detected in native gelatin-containing polyacrylamide gels, have their activities increased during nitrogen-limited growth of the mycelium. ScPrB, a metallo-endoprotease, appears to have the greatest nitrogen stress-induced increase in activity of all of these enzymes. Quantifying ScPrB has proven difficult because no artificial chromogenic substrate has been found. In addition, it is poorly resolved from another highly active protease, ScPrA, in native gelatin-containing gels. We have developed a method using SDS gelatin-containing polyacrylamide gels for resolving ScPrB from ScPrA and for quantifying its activity by densitometry. This method was used to assess the intramycelial location of ScPrB induction after the transfer of exponentially growing colonies to nitrogen deprivation conditions. By all analyses (proportional, normalized to fresh weight, and normalized to protein), the increase in ScPrB activity was found to occur exclusively in midsections of the growing mycelium, whereas ScPrB activity was found to be decreased or unchanged in the centers of colonies and in colony margins. This implies that proteolysis mediated by ScPrB may supply translocatable amino acids only from the region directly behind the growing hyphal apices.