The prognostically important 1;19 chromosomal translocation can alter the E2A gene on chromosome 19p13 in childhood B-cell precursor acute lymphoblastic leukemia (ALL), leading to formation of a fusion gene (E2A-PBX1) that encodes a hybrid transcription factor with oncogenic potential. It is not known whether this molecular alteration is a uniform consequence of the t(1;19) or is restricted to translocation events within specific immunologic subtypes of the disease. Therefore, we studied leukemic cells from 25 cases of B-cell precursor ALL, with or without evidence of cytoplasmic Ig mu heavy chains (cIg); 17 cases had the t(1;19) by cytogenetic analysis. Leukemic cell DNA samples were analyzed by Southern blotting to detect alterations within the E2A genomic locus; a polymerase chain reaction assay was used to identify expression of chimeric E2A-pbx1 transcripts in leukemic cell RNA; and immunoblotting with anti-Pbx1 antibodies was used to detect hybrid E2A-Pbx1 proteins. Of 11 cases of cIg+ ALL with the t(1;19), 10 had E2A-pbx1 chimeric transcripts with identical junctions and a characteristic set of E2A-Pbx1 hybrid proteins. Each of these cases had E2A gene rearrangements, including the one in which fusion transcripts were not detected. By contrast, none of the six cases of t(1;19)-positive, cIg- ALL had evidence of rearranged E2A genomic restriction fragments, detectable E2A-pbx1 chimeric transcripts, or hybrid E2A-Pbx1 proteins. Typical chimeric E2A-pbx1 transcripts and proteins were detected in one of eight cIg+ leukemias in which the t(1;19) was not identified by cytogenetic analysis, emphasizing the increased sensitivity of molecular analysis for detection of this abnormality. We conclude that the molecular breakpoints in cases of cIg- B-cell precursor ALL with the t(1;19) differ from those in cIg+ cases with this translocation. Leukemias that express hybrid oncoproteins such as E2A-Pbx1 or Bcr-Abl have had a poor prognosis in most studies. Thus, molecular techniques to detect fusion genes and their aberrant products should allow more timely and appropriate treatment of these aggressive subtypes of the disease.