We investigated the possible role of metal proteinase on the fruit-body formation of Hypsizygus marmoreus. The addition of a specific metal proteinase inhibitor, phosphoramidon, to the culture medium (10μg/ml) completely inhibited fruit-body formation. Metal proteinase activity in both the medium and the mycelia of this fungus increased markedly during vegetative mycelial growth, and activity was maximal 25 days after inoculation. When phosphoramidon was added to the culture medium during vegetative mycelial growth, the metal proteinase activity in the mycelium decreased to 56% of the control (without inhibitor) level. Isoelectric focusing analysis showed that two kinds of metal proteinases with a pl of 7.7 and 8.4, respectively, were obtained from 29-day-old mycelia. Uptake of phosphoramidon into the mycelia was confirmed as the result of inhibition of thermolysin activity by the mycelial extracts. The degree of inhibitor uptake into mycelia was about 2.0% and was independent of the initial concentration of the inhibitor administered. The addition of peptone and amino acids to medium treated with phosphoramidon resulted in fruit-body dry weight yields that were about 50% that of the control.