Antisera were raised in rabbits against kringle 4, one of the five homologous domains of human plasminogen, and one which contains a site that binds lysine. A double antibody radioimmunoassay was developed for kringle 4 and used for cross-reactivity studies with plasminogen, kringle 1 + 2 + 3, kringle 5 + light chain, and the plasmin-antiplasmin complex, as competitors of either 125I-kringle 4 or 125I-Lys-plasminogen for binding to anti-kringle 4 antisera. Lys- and Glu-plasminogen caused 50% inhibition of anti-kringle 4 binding to 125I-kringle 4 at ratios of 4 and 20 mol/mol (competitor/kringle 4), respectively. The plasmin-antiplasmin complex behaved similarly to Glu-plasminogen. Kringle 1 + 2 + 3 inhibited at a ratio of about greater than 1000 mol/mol. Our results suggest that most of the surface antigenic sites of kringle 4 are exposed on Lys-plasminogen, Glu-plasminogen, and the plasmin-antiplasmin complex. There may be weak antigenic homology between kringle 4 and kringle 1 + 2 + 3, but there is little or no homology with kringle 5 + light chain and prothrombin fragments 1 and 2. Both 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexanecarboxylic acid can cause a maximum of 30% inhibition, with the latter being a much better inhibitor. Our results suggest that part of the antibody population is directed against a region of kringle 4 containing a lysine-binding site.