In this manuscript, we present a new bifunctional support containing epoxide and thiol-reactive groups for its use in protein immobilization. In a first step, the proteins are reversibly immobilized by reaction of its thiol groups with the thiol-reactive groups of the support under mild experimental conditions (pH 7.0, 24 degreesC). Then, the remaining epoxides of the support can form irreversible bonds with nucleophile surface groups of the already immobilized protein in a rapid way. The partial derivatization of EP-Sepabeads (a commercial matrix containing 120 mumol epoxy groups/g drained support) was optimized, using dithiotreitol (DTT) as thiolating agent. It was possible to achieve a partial thiolation of the support proportional to the concentration of DTT used (3, 8, and 15 mumol SH groups/g wet support). The remaining epoxide content after the thiolation treatment was high (e.g., nearly 70% for the highest thiolation degree). High immobilization yields were obtained for the three model enzymes selected (60% for Penicillin G acylase, 65% and 100% for K. lactis and E. coli beta-galactosidase, respectively). In all cases, no significant immobilization onto an unmodified epoxy support was found, thus demonstrating that the first step of attachment takes place through thiol-disulfide exchange reactions. In the case of the bifunctional support, progressive formation of enzyme-support attachments involving the epoxy groups was showed by the irreversible covalent attachment of the proteins on the support. The promotion of this multipoint covalent immobilization required long incubation periods at basic pH values.