A protein-binding radioimmunoassay (RIA) of rat low molecular weight (LMW) kininogen with the following characteristics has been developed: sensitivity, 2.5 ng/tube; inter-assay coefficient of variation, 12.4% (N = 28); and intra-assay coefficient of variation, 9.4% (N = 11). The new assay correlated (r = 1) with the determination of kinin equivalence of kininogen after trypsinization. The cross-reactivity of rabbit anti-rat LMW kininogen antibody was 2.5% with bovine LMW kininogen, 5.8% with rat plasma high molecular weight (HMW) kininogen, and none with kinin. Although the antibody appears to partially recognize des-kinin-kininogen, the low degree of cross-reactivity and the extremely low levels of kinin-free-kininogen allow accurate determination of total LMW kininogen in rat urines. The LMW kininogen formed 20% kinins with salivary kallikrein when compared with trypsin, suggesting that the preparation consists of both K- and T-kininogens (K = kallikrein susceptible; T = trypsin susceptible). The newly developed protein-binding RIA recognizes LMW kininogen of rat urine which consists of both K- and T-kininogens.