Two antisera used in the radioimmunoassay for human fibrinopeptide A (FPA) which appear to have different immunochemical specificities have been tested for cross-reactivity with fibrinogen and with three fragments of fibrinogen which contain the FPA sequence. These fragments were the three-chain, NH2-terminal disulfide knot (N-DSK) produced by CNBr cleavage of fibrinogen, the reduced, carboxymethyl Aalpha chain portion of the N-DSK, and fragment E produced by plasmin digestion of fibrinogen. One antiserum (R-2) showed high specificity for free FPA and less than 2% cross-reactivity with fibrinogen or the FPA-containing fragments. The other antiserum (R-33) possessed a much higher degree of cross-reactivity with the FPA-containing fragments. Synthetic and native fibrinopeptides were found to be indistinguishable in the assay system with either antiserum. As a result of these studies, an hypothesis has been developed concerning the nature of the antigenic determinants on FPA which favor measurement of free FPA and limit cross-reactivity with larger, FPA-containing peptides.