Allelic exclusion normally results in the expression of only one light and one heavy chain gene. However, some hybridomas have been reported to express two different heavy chain genes. Here we report that the IgG3 hybridoma 4H3.C8B expresses both kappa and lambda light chains. 4H3.C8B was originally recovered by screening for antibody binding to Cryptococcus neoformans polysaccharide antigen and characterized as gamma 3 lambda. The gamma 3 lambda but not the gamma 3 kappa binds to polysaccharide antigen. Some of the antibody molecules were heterodimers composed of both lambda and kappa. IgG1 and IgG2b isotype switch variants were identified and isolated by the technique of sib selection, using the ELISA spot assay. Like the parent IgG3 line, the switch variants continued to express both kappa and lambda light chains but only the lambda-containing antibodies bound to the antigen. Our experience suggests that hybridomas recovered by assays that are antigen dependent should also be tested for the expression of other isotypes and light chains in a non-antigen-binding immunoassay.