BALB/c mice were exposed simultaneously to three nonrelated immunogens, myelin basic protein, uridyl-galactosyl transferase, and tissue obtained from a formalin-fixed, paraffin-embedded block containing a pilomatrixoma. Standard hybridoma techniques were used and antibody generation assayed using an unlabelled antibody biotin-avidin method with sections of human cerebellum, liver, and pilomatrixoma as the substrates. Using the above assay, clones were selected that secreted antibodies with selective specificities for each of the immunogens. By ELISA assay, the monoclonal antibodies reacting with cerebellum also reacted to the myelin basic protein preparation used as immunogen and the monoclonal antibody reacting to hepatocytes bound to the preparation of uridyl-galactosyl transferase used as immunogen. Our data suggests that the generation of monoclonal antibodies with a variety of diagnostic applications can be obtained from a single fusion following immunization with multiple nonrelated antigens, requiring considerably less laboratory cost and effort than would be required to obtain similar monoclonal antibodies in separate fusions.