Hybridomas producing antihuman light chain monoclonal antibodies (MoAbs) were derived from fusion of SP2/O mouse myeloma cells with splenic lymphocytes from mice repeatedly immunized with purified Kand κ-type Bence Jones proteins representative of the major Vκ, (VκI, VκII, VκIII, VκIV) and Vλ (VλI, VλII/V, VλIII, VλIV, VλVI,) subgroups or gene families. Monoclonal antibodies were obtained that had specificity for constant-region (CI) determinants common to all κ or λ light chains (Cκ and Cλ, respectively) as well as for variable-region (VL) epitopes unique to each of the Vκ or Vλ subgroups. The capability of these reagents to recognize CI and VL determinants on monoclonal immunoglobulin (Ig) molecules was demonstrated in fluid-phase antigen-capturing enzymelinked immunosorbent assay (ELISA), solid-phase ELISA, and immunoblotting. In addition, these antilight chain MoAbs were used to establish immunocytochemically the κ or λ type and VL-subgroup nature of light chains expressed by the cytoplasmic Ig of monoclonal plasma cell and surface Ig of B-lymphocyte populations, respectively. These antibodies facilitated the immunohistochemical detection and characterization of light-chain-associatcd amyloid (AL amyloid) and other types of light-chain-related tissue deposits. Furthermore, the unti-CL-specific MoAbs were used to measure serum and urinary Igκ and Igλ concentrations. Quantification of Bence Jones protein excretion, even in the presence of other urinary proteins, was possible using the highly sensitive anti-Cκ and anti-Cλ MoAbs reactive only with free light chains. The ability to identify and characterize, through the use of these antihuman light chain MoAbs, light-chain-related epitopes at the protein, cellular, and tissue level has clinical importance in the diagnosis and treatment of patients with monoclonal plasma cell and related B-cell immunoproliferative diseases.