t(12;21)(p13;q22)[ETV6-RUNX1] is the most common chromosomal translocation in childhood acute lymphoblastic leukemia, and it can often be backtracked to Guthrie cards supporting prenatal initiation and high levels of circulating t(12;21)-positive cells at birth. To explore the prevalence of ETV6-RUNX1-positive cells in healthy neonates, mononuclear cells from 1417 umbilical cord blood samples were isolated within 24 hours from birth and subsequently screened for ETV6-RUNX1 transcripts using a highly sensitive real-time reverse transcription polymerase chain reaction assay. In first-run polymerase chain reaction, 14 samples were positive at levels below 10(-5), of which specific hybridization reflecting the relevant genetic region was positive in 9 cases. Repeated analyses using stored mRNA and flowcytometric sorting of a CD19(+), CD8(+), and CD19(-)/CD8(-) subpopulations from cryopreserved mononuclear cells from the same cord blood samples (mean sorted: 18 × 10(6) cells) revealed no positive findings, which demonstrates that the level and/or frequency of ETV6-RUNX1-positive cells is markedly lower than suggested in previous studies.