The mouse monoclonal antibodies (mAb), 3B5 and 4B5, which recognize rabbit kappa light chains bearing the b5 allotype, were produced from separate fusions. The specificity of the mAbs was determined by solid-phase inhibition radioimmunoassay. Nonimmune sera of 15 b5b5 rabbits of various heavy chain haplotypes inhibited the binding of both mAbs to b5 IgG, whereas 20 sera from rabbits not expressing the b5 allotype were not inhibitory. In addition, the binding of both mAbs was inhibited by purified b5 light chains, but not by b4 light chains. The b5 epitope recognized by the mAbs was shown by sequential precipitation to be present on all b5-bearing molecules that are defined by an alloantiserum produced in a b4b4 rabbit. Antibody 4B5 forms strong precipitin bands with b5 serum and Ig in gel diffusion assays. An anomalous reaction of nonidentity was observed when mAb was compared to rabbit anti-b5 antiserum and a hypothesis to explain this phenomenon is proposed.