Type II restriction endonucleases Bam HI and Eco RI were covalently coupled to Sepharose. These insolubilized enzymes generated fragment patterns for several viral DNAs identical to those produced by the respective free enzymes. Conditions for optimal activity were similar for both bound and unbound forms of the enzymes. Insolubilization improved thermal stability of Bam HI and Eco RI. The bound enzyme can be recovered from reaction mixtures and reused several times. Upon storage at 4°C, coupled endonucleases remained stable for several months.