The primary goal of this study was to develop a method for evaluating fibrin-specific antibodies as thrombus detecting agents. The apparatus and assay conditions were chosen by testing antibody 64C5, which binds to the amino terminus of the fibrin beta chain, for its ability to bind to human blood clots. Using 125I-labeled antibody 64C5, the effects of antibody concentration, clot shape, clot mass, temperature, and flow rate were tested. Increased antibody binding was observed when antibody concentration, clot mass and temperature were increased. Under one set of conditions, six 125I-labeled monoclonal antifibrin antibodies (four specific for the beta chain, two specific for the alpha chain) were tested for their binding to retracted clots of human blood. Two radioiodinated antidigoxin antibodies were used as a control. Beta chain-specific antibody 59D8, which provided the highest level of binding to clot, bound 14-fold better than the control antidigoxin antibody. Neither alpha chain-specific antibody bound to clotted blood. To examine the in vivo relevance of the in vitro binding to clots, the uptake of antibody 64C5 was assessed for its binding to human fibrin clotted within the jugular vein of a rabbit. The correlation coefficient between in vitro and in vivo uptake as a function of clot weight was calculated to be 0.91. Thus, in vitro binding of monoclonal antifibrin antibodies to human blood clots was judged to be a realistic method for the comparison and selection of an ideal antifibrin antibody for detailed in vivo testing.