Two activated matrices have been developed to determine whether immobilization chemistry can be used to orient proteins on a support. Restriction endonuclease EcoRI from Escherichia coli RY13 (E.C.3.1.23.13) was used as a model in these studies. Thiol-activated Sephadex G-10 was used to couple the EcoRI endonuclease through its free sulfhydryl, while amino-activated Sephadex G-10 was used to couple it randomly through its free carboxyl groups. To determine whether the enzyme was immobilized randomly or specifically, both lower and higher molecular weight substrates were used. The polymerase chain reaction amplified multiplied cloning site region of pBluescript KS obtained using T3 and T7 primers was considered as the small substrate. The plasmid SP64 containing firefly luciferase gene was the large substrate. Immobilized EcoRI preparations were characterized with respect to repeated usage and storage stability. The EcoRI immobilized on thiopropyl-Sepharose 4B could be stored for over 14 days at 4 degrees C without observable loss of activity. In an independent experiment the same gel was used thrice repeatedly without any discernible loss of activity.