An 81-year-old woman, who presented with sudden episodes of spontaneous bleeding, was found to have a specific inhibitor of factor XIII. Her fibrin clots had approximately 70% gamma-gamma and no alpha polymer formation, under conditions where normal fibrin was fully cross-linked; the patient's clots were soluble in urea or monochloroacetic acid. Factor XIII activity in her plasma was 24%, measured by the dansylcadaverine incorporation assay. When mixed with normal plasma, the patient's plasma inhibited fibrin cross-linking; however, in mixtures of patient and normal plasma, there was no inhibition of factor XIII activity when assayed by the incorporation of dansylcadaverine into casein. Thus, this inhibitor was active against fibrin cross-linking but not against ligation of small molecules to casein. Consequently, gel electrophoresis of reduced, sodium dodecyl sulfate-solubilized fibrin clots was a simple, quantitative method that was used to measure inhibitor activity. This inhibitor is unique and has been designated inhibitor New Haven. It was neutralized by anti-IgG and anti-kappa. It did not inhibit the activation of factor XIII but did inhibit fibrin cross-linking. There was complex formation between the inhibitor and activated factor XIII (A', A*) but not between A2 or fibrinogen. Only A', A* and the 56-Kd fragment bound to affinity columns made with this IgG. The inhibitor significantly decreased the binding of A', A* to fibrin clots. These data indicate that the epitope for this inhibitor is in a fibrin binding site. It is hidden in the zymogen and expressed on A' and A*, indicating that the conformational change occurring with the cleavage of the activation peptide is sufficient to expose the fibrin binding site.