The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the gamma-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B, was highly effective in binding EcoRI from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRI immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear lambda-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of EcoRI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65 degrees C. It was also possible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRI to the antibody support.