Immunoblotting of blood coagulation Factor XII and plasma prekallikrein in whole plasma was performed using radiolabeled antigen for detection. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of plasma and transfer to nitrocellulose sheets, the blots were first reacted with polyclonal goat anti-Factor XII or anti-prekallikrein antisera and then with 125I-Factor XII or 125I-prekallikrein, respectively. A major advantage of using radiolabeled antigen rather than radiolabeled secondary antibody was enhanced specificity of immunodetection of these antigens in plasma. This procedure was sensitive to approximately 0.3 ng of either Factor XII or prekallikrein antigen and was useful for detection of Factor XII cleavage fragments in contact activated plasma. Radiolabeled antigen overlay may improve the specificity of immunoblotting of trace antigens in any complex mixtures.