Ectomycorrhizal fungi have been shown to survive sub-zero temperatures in axenic culture and in the field. However, the physiological basis for resistance to freezing is poorly understood. In order to survive freezing, mycelia must synthesise compounds that protect the cells from frost damage, and certain fungal-specific soluble carbohydrates have been implicated in this role. Tissue concentrations of arabitol, mannitol and trehalose were measured in axenic cultures of eight Hebeloma strains of arctic and temperate origin grown at 22, 12, 6 and 2 degrees C. In a separate experiment, mycelia were frozen to -5 degrees C after pre-conditioning at either 2 degrees C or 22 degrees C. For some, especially temperate strains, there was a clear increase in specific soluble carbohydrates at lower growth temperatures. Trehalose and mannitol were present in all strains and the highest concentrations (close to 2.5% and 0.5% dry wt.) were recorded only after a cold period. Arabitol was found in four strains only when grown at low temperature. Cold pre-conditioning enhanced recovery of mycelia following freezing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryoprotection. The results are discussed in an ecological context with regard to mycelial overwintering in soil.