Hybridoma technology has been used for the production of murine monoclonal antibodies to bovine coagulation Factor V and its thrombin-activated product, Factor Va. Hybrid cell cultures were assayed for the production of anti-Factor V and anti-Factor Va antibodies by a solid-phase radioimmunoassay. Antibody-producing cell lines were selected, cloned and grown as ascites tumors. Gel filtration chromatography (Ultrogel AcA34) and affinity chromatography (protein A-Sepharose) were used to isolate the monoclonal immunoglobulins from the ascites fluids. Thirteen monoclonal antibodies have been characterized with respect to their binding to Factor V and Factor Va and their effect on cofactor bioactivity. Six of these thirteen antibodies react with both Factor V and Factor Va. One of these antibodies is strongly inhibitory, while a second antibody is only moderately inhibitory. The antibody produced by another cell line binds Factor V but not Factor Va and is not inhibitory. The remaining six cell lines each produce an antibody that reacts preferentially with Factor Va, and each of these antibodies is inhibitory to some extent. Both a radioimmunoassay and light scattering have been used to study the interaction of the immunoglobulins with Factor V and Factor Va. The light scattering technique has proven useful to study the interaction of isolated antibodies and antigens and permits the determination of interaction stoichiometries. Each of the interactions studied was characterized by a stoichiometry of two antigens per antibody. These monospecific immunochemical reagents will be useful in the study of structure and function relationships of Factor V, Factor Va and activation fragments.