The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.