The edible mushroom, Agaricus bisporus, when cultivated commercially derives its nitrogen from the proteins present in compost, much of which is firmly bound to the lignin-humic complex. The proteinases involved in mobilization of nitrogen were studied in both the filtrates of liquid culture and mushroom compost. Proteinase activities were detected in liquid culture filtrates with a range of substrate specificities. One activity with chymotrypsin-like specificity was found by Western blotting to be the 27 kDa serine proteinase previously purified from senescent sporophores. Serine proteinase activity and level in the filtrates rapidly increased during mycelial growth on liquid medium containing humic fraction (extracted from mushroom compost) as sole nitrogen source. Activity and level remained low in filtrates of cultures grown on glutamate or casein. Non-denaturing gel analysis revealed the serine proteinase to be the most prominent proteolytic activity in compost filtrates. Regulation of the activity in compost was indicated by a doubling during the early stages of sporophore growth.