Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through κ and δ light (L) chain genes. To determine whether the predicted κ → λ, hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface μ/λ, by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the μ/λ-expressing cell lines contained both μ alleles in germline configuration, and synthesis/expression of conventional λ L chains was directly proven by immunoprecipitationAodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional λL chain gene rearrangements without rearranging or deleting either κ allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface κ and λ L chains associated with μ H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both κ alleles rearranged, and a single λ rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed κ and λ L chains. Multiparameter flow cytometry was used to demonstrate the existence of κ+/λ+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface κ and λ refutes the widely accepted concept that expression of a single L chain isotype is immutable. The κ+/λ+ cells may represent transients undergoing L chain isotype switching.