Although the advances offered by the development of monoclonal antibody techniques have revolutionized the specificity, uniformity, and quantity of antibodies, there remain many circumstances in which polyclonal antibodies are more desirable than monoclonal antibodies. Production of polyclonal antisera takes less time and effort than production of monoclonal antibodies, requires relatively simple and readily available equipment, and produces reagents that can be used for immunoprecipitation, immunoblotting, and enzyme-linked immunosorbent assays (ELISAs). The Basic Protocol and Alternate Protocol in this unit describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. The Support Protocol presents a method for preparing serum from blood.