The t(1;19)(q23;p13) translocation involving the E2A gene on chromosome 19p13.3 is a nonrandom translocation that is often seen in childhood pre-B-cell acute lymphoblastic leukemia (ALL). However, recent studies have demonstrated the presence of immunophenotypic and molecular heterogeneity among patients with the cytogenetically identical chromosome translocation. Here we report a novel pre-B ALL cell line, TS-2, with t(1;19) translocation not involving the E2A gene. The breakpoint of t(1;19) in TS-2 was demonstrated to be at 19p13.3, a region indistinguishable from the locus of the E2A gene, by cytogenetic study and fluorescence in situ hybridization. However, rearrangement of the E2A gene was not detected in TS-2 by Southern blot analysis. Moreover, the expressions of PBX1 or E2A/PBX1 fusion genes were not detected by an extensive study with Northern blot analysis and reverse transcription–polymerase chain reaction. These findings suggest that TS-2 may have a genetic abnormality involving uncharacterized gene(s) at 19p13.3 distinct from the E2A gene and, therefore, may be useful for investigating the heterogeneity of molecular pathogenesis in leukemias with t(1;19)(q23;p13) translocation.