Antibody populations against high- and low-molecular weight urokinases (HUK and LUK) were purified by passage of rabbit antiserum immunoglobulin fractions made against HUK and LUK through HUK- or LUK-bound Sepharose 4B (Pharmacia AB, Uppsala, Sweden) gels, respectively. Radioimmunoassays were then set up with these antibody populations. The characteristics of these radioimmunoassays were studied by competition of different antigens against radiolabeled HUK or LUK for these antibodies. We found that the anti-HUK antibody population recognizes epitopes on the light chain of HUK, most of which are situated outside of the urokinase-receptor binding sequence of amino acids. The anti-LUK antibody population probably recognizes a region on LUK distinct from its catalytic site. This region is also present in HUK but is altered in single-chain urokinase, suggesting that a conformation change takes place when single-chain urokinase is converted to HUK. The complexes of HUK and LUK with plasminogen activator inhibitor 1 or 2 could compete in either radioimmunoassay, with different and diminished efficiencies. Taking advantage of this result, we devised a procedure to measure the concentrations of various urokinase species in human urine. We found that freshly voided urine contained substantial amounts of free HUK, some HUK- and LUK-plasminogen activator inhibitor complexes, and a small amount of single-chain urokinase. By using a combination of radioimmunoassay and zymography, we found that human plasma contains mostly HUK-plasminogen activator inhibitor 1 complexes, a small amount of single-chain urokinase or HUK, and no LUK or LUK-inhibitor complexes.