Preliminary observations have indicated that both heavy and light chains of monoclonal immunoglobulin exhibit diagnostically useful microheterogeneity patterns when examined with high resolution two-dimensional electrophoresis (2DE) and silver staining (Clin Chem 1990; 36: 993). The present study is a survey of the distribution and types of monoclonal light chain isoforms as observed with 2DE in a group of 68 sera and urine specimens from a cohort of 41 patients with monoclonal gammopathy. The light chain patterns were patient-specific and varied in both the type (kappa, lambda, charge or mass or both) and the number of isoforms present (one to six). A large proportion (31 of 41 cases, 75.6%) exhibited significant microheterogeneity, i.e., distribution of light-chains among two or more isoforms. Charge-based microheterogeneity alone occurred in 17 cases and both charge and mass isoforms were observed in 14 cases. The distribution of charge isoforms of the light chains corresponded qualitatively to the densitometric profiles of agarose gel and immunofixation electrophoresis studies. There was no correlation between heavy chain type and light chain pattern. A carbamylation assay was used to demonstrate that the chemical charge differences between the isoforms were unitary. Ascertainment of this light-chain microheterogeneity has not only proved useful in resolving some diagnostic immunoglobulin typing dilemmas but also is germane to understanding the molecular basis of neoplastic B-cell paraprotein metabolism.