Subculture is the most common method for preservation fungi, but has a disadvantage of accumulation of sponta- neous mutations during the repeated subculture. To reduce the subculture frequency, the effect of preservation period at 4ºC in a slant culture was examined on the mycelia growth and lignocellulolytic enzyme activities of various basidiomycetes. Mushrooms, including , , , , , , and 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.