Two novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) sister cell lines, designated NALM-36 and NALM-37, were established from the peripheral blood (at diagnosis) and bone marrow (at relapse) of a 37-year-old woman with ALL. Immunophenotyping showed BCP type III pre-B cell characteristics including TdT, CD10, CD19, CD22, CD79a and HLA class II. T cell and myeloid-associated antigens tested were negative except CD5 which was 100% positive for both cell lines. The surrogate light chains lambda5 and VpreB were positive for both cell lines. Cytogenetic analysis of NALM-36 revealed an abnormal karyotype with 46, XX, add(1)(q?42), -14, +mar. Southern blot analysis of the immunoglobulin (Ig) genes status of NALM-36 at 10 months after establishment showed germ line configuration of the kappa light chain gene, and rearrangement of the lambda light and mu heavy chain genes. At 16 months we detected a phenotypic shift of Ig chain protein expression from a BCP-III pre-B cell phenotype to a BCP-IV mature B cell phenotype, with kappa and lambda double Ig light chain and mu heavy chain expression, both on the cell surface and in the cytoplasm. We designated this subline as NALM-36KL. Authenticity of the NALM-36KL, NALM-36 and NALM-37 cell lines was demonstrated by DNA fingerprinting. The extensive characterization of the sister cell lines suggests that these three novel cell lines, derived from a single patient, may represent unique and relevant in vitro model systems for BCP-type leukemia cells. They may provide useful models and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.