The t(1;19)(q23;p13) translocation occurs in approximately 5% of B- precursor acute lymphoblastic leukemias (ALLs) occurring in children. Its presence has been associated with a poor prognosis, which may be overcome with more intensive therapy. Although leukemic cells from cases of t(1;19)-ALL frequently express cytoplasmic mu heavy chains, their complete antigenic profile remains undefined. Among 697 consecutive cases of B-precursor ALL with complete phenotypic studies using a panel of monoclonal antibodies, 22 cases were found to carry the t(1;19). Twenty of 22 cases had an identical, complex phenotype characterized by homogeneous expression of CD19, CD10, and CD9; complete absence of CD34; and at least partial absence of CD20. Overall, this phenotype was seen in only 8.0% (56 of 697) of childhood B-precursor ALL. One of the two remaining t(1;19)-carrying cases conformed to this phenotype, but was lacking data for CD9. The other case differed by virtue of expression of CD34 and was also hyperdiploid with 55 chromosomes. Molecular studies showed E2A-PBX1 abnormalities in all examined cases (12 of 12) with the t(1;19), including the case lacking CD9 data. In contrast, no E2A-PBX1 abnormalities were detected in the sole t(1;19)-ALL with CD34 expression. Seventeen cases with the characteristic phenotype and uninformative cytogenetics were also molecularly analyzed and 5 of 17 (including 4 of 8 with unsatisfactory cytogenetics and 1 of 9 with a normal karyotype) contained E2A gene rearrangements and E2A-PBX1 fusion mRNAs. Our results show that all cases of t(1;19)-ALL with concomitant E2A-PBX1 fusion invariably express a characteristic but uncommon profile of surface antigens. These observations suggest that selective molecular analysis of a small subset of patients (those with uninformative cytogenetics and the characteristic phenotype) can identify a significant number of additional cases of ALL with E2A-PBX1 fusion that might benefit from more intensive therapy.