This experiment was undertaken to investigate proper conditions for protoplast formation from Lyophyllum ulmarium. Combination of Novozym 234, ${eta}-Glucuronidase$ and ${eta}-D-Glucanase$ with 0.6 M Sucrose was the most effective for isolation of protoplasts. The optimal reaction time of mycelium with the lytic mixture was 3 hrs in shaking condition at 120 strokes $min-^1$. When the mycelium of L. ulmarium was cultured for 6 days on yeast glucose agar medium at $25^{circ}C$, the formation of protoplasts was effective. The yeast glucose agar medium stabilized with 0.6 M sucrose was the most effective for reversion of protoplasts.