A murine hybridoma monoclonal line-secreted antibody (C3G5) against the light chain of human high molecular weight kininogen (HMWK), which consisted of gamma-1 kappa isotype, was largely composed of 190 kd molecules, and gave an optimal reaction when used in an equimolar concentration with HMWK. It did not influence the initial digestion of HMWK or the amount of kinin released by kallikrein. Pretreatment of HMWK with C3G5 antibodies did not augment or inhibit its coagulant properties, nor did pretreatment interfere with its adsorption on kaolin or on the ion-exchange resin diethylaminoethyl (DEAE) Sephadex A-50. The epitope that reacted with this antibody was localized to a portion of the light chain within 6 kd of the carboxy-terminus of the molecule near the single cysteine of this chain, at residue 614. This monoclonal antibody reacted with an epitope that was stable during early plasmin digestion. The amounts of antigens reactive with a polyclonal antibody to the light chain were increased during the same period of plasmin digestion. Prolonged plasmin digestion reduced the amounts of both types of light chain antigens. During kallikrein digestion, the amount of epitope reactive with C3G5 antibody was unaffected, whereas the concentration of antigens detected with polyclonal anti-light chain antibodies was increased throughout digestion. Therefore, light chain antigens reactive with the polyclonal antibody are probably in the interior of the uncleaved molecule and become exposed after initial cleavage, which probably facilitates changes in the conformation of the molecule. The epitope reactive with monoclonal antibodies is probably on the surface of the uncleaved molecule.