Over a period of 5 years, an isolated light chain (kappa = 9, lambda = 12) was detected in 21 sera by immunofixation electrophoresis. Further analysis with anti-delta- and anti-epsilon-specific antisera identified four delta heavy chains, all associated with a lambda light chain, and no epsilon heavy chains. For evaluation of the role of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in the diagnosis of IgD paraproteins, as a possible alternative or complement to immunofixation, IgD paraproteins were retrospectively analyzed by 2D-PAGE. Delta Heavy chains migrated to gel areas clearly distinguishable from other heavy chains alpha, gamma, or mu, and in a wide range of isoelectric points (pI: 5.4-8). In one serum, the monoclonal delta chain had a pI range comparable to that of albumin and was undetectable. However, all four delta chains were easily identified when analyzed from affinity-purified immunoglobulin fractions. These observations showed the following: 1) IgD paraproteins are not rare among apparently isolated monoclonal light chains detected by routine immunofixation, strongly confirming the need for further analysis with anti-delta antisera, before assumption of a light-chain disease. 2) 2D-PAGE analysis of affinity-purified immunoglobulin fractions allowed correct identification of IgD monoclonal gammopathies in all cases. 3) However, although 2D-PAGE analysis is now easy to perform, well standardized, and highly sensitive, this technique remains time-consuming and expensive, and does not appear suitable for routine practice as a first-line diagnostic procedure. 2D-PAGE should find its place as a complement to immunofixation and in the definitive demonstration, in selected ambiguous cases, of the clonal pattern of a suspected gammopathy at immunofixation.