A strong fibrin-specific fibrinolytic enzyme was produced by liquid culture of Coprinus comatus YY-20. Soybean cake powder and sucrose as substrates resulted in the highest fibrinolytic activity. The optimal temperature for the production of the enzyme was found to be 21-25°C in the optimal medium (Soybean cake powder 50 g/L, sucrose 20 g/L). The optimal initial pH of the medium was found to be at the range of 5-8. The maximum fibrinolytic activity was observed at the stationary phase of cell growth. These results suggest that the culture liquid of Coprinus comatus YY-20 has potential as a source of physiologically active fibrinolytic enzymes.