Subculture is the most common method for preservation fungi, but has a disadvantage of accumulation of spontaneous mutations during the repeated subculture. To reduce the subculture frequency, the effect of preservation period at 4 oC in a slant culture was examined on the mycelia growth and lignocellulolytic enzyme activities of various basidiomycetes. Mushrooms, including Stereum ostrea, Coprinellus micaeus, Trametes versicolor, Hypholoma fasciculare, Wolfiporia extensa, Pleurotus pulmonarius, Piptoporus betulinus and Ganoderma applanatum were not affected by the preservation period more than two years, indicating that they can be maintained by subculture every two years. Some other tested fungal strains showed a significant decrease in both viability and enzyme activity when they were maintained for two years, suggesting that they should be subcultured at least once in a year. A little correlation was found between the recovery of mycelial growth and extracellular enzyme activity. In conclusion, mycelial activity and enzyme activity according to storage period is expected to be a way of deciding on subculture times for fungal preservation.