The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at -84 degrees C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 x 10(4) zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at -84 degrees C.