The methylviologen-dependent hydrogenase of Anabaena sp. CH3 is unstable at 4 degrees C and in air. All the purifying procedures were carried out at 25 degrees C and under argon atmosphere. The enzyme was partially purified by the following steps: heat treatment, DEAE-cellulose ion-exchange chromatography and gel filtration on TSK-Fractogel. Experimental results indicated that the heat-treatment procedure was beneficial for the separation of the enzyme from phycobiliproteins.