We have investigated the karyotypes and markers of B lymphocyte differentiation in two leukemic cell lines, NALM‐6‐B and NALM‐6‐MI, both derived from a patient with non‐T, non‐B acute lymphoblastic leukemia. Both possess characteristics of thymus‐independent, bursalequivalent (B) lymphocytes. By means of immuno‐fluorescence techniques, NALM‐6‐B was shown to possess 47% surface membrane immunoglobulin‐positive (Slg+) cells, while NALM‐6‐MI is surface membrane imunoglobulin‐negative (Slg−), but does possess cytoplasmic immunoglobulin M (ClgM+) in more than 90% of the cells. The Slg− ClgM+ phenotype, coupled with morphologic features, is consistent with NALM‐6‐MI being arrested at a stage early in B‐lymphocyte differentiation (a “pre‐B” cell). The predominant banded chromosome karyotype of NALM‐6‐B is pseudodiploid with a translocation of chromosomal material from the long arm of a chromosome 5 to the short arm of a chromosome 12 (5q−/12p+) and a marker Y chromosome. NALM‐6‐MI possesses the same marker Y chromosome and a deletion of the long arm of chromosome 5 (5q−) since in most cells material missing from chromosome 5 does not appear on the short arm of chromosome 12. Within the limits of resolution available with the banding technique, no other karyotypic differences are observed in the two cell lines. The history of the two cell lines, the karyotypic analysis, and the pattern of immunoglobulin markers indicate the possible clonal evolution of NALM‐6‐MI from NALM‐6‐B and implicate a portion of the long arm of chromosome 5 in lymphoid differentiation of these leukemic cell lines.