An enzyme-linked immunospecific assay (ELISA) was developed to screen monoclonal antibody to human thyroxine-binding globulin (TBG). The assay is based on the absorption of TBG from human pooled serum by rabbit polyclonal anti-TBG antibody coated on a microtiter plate and subsequent binding of monoclonal antibody to the absorbed antigen. Monoclonal antibody which binds TBG can be detected by peroxidase labeled anti-mouse IgG. From the examination with two different rabbit polyclonal anti-TBG antibodies, it was demonstrated that both purified IgG fraction and whole serum could be used as coating materials. It was not necessary to use purified TBG to react with anti-TBG antibody coated on the microtiter plate. When eight commercially available microtiter ELISA plates were tested in the assay, only 3 gave satisfactory results. The sensitivity of the assay was comparable with that of the conventional immunoprecipitation method using 125I-TBG and formalin fixed Staphylococcus aureus (Kowan strain) as an immunoabsorbent. The ELISA method could detect antibody activity in 0.032 microliter of medium obtained from a 3 day culture of confluent hybridoma cells. It is possible to store the antibody-antigen complexed microtiter plate for more than 2 weeks at 4 C. This makes possible rapid screening of monoclonal antibody.