A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.