Involvement of the ETV6 gene, located at 12p13, has been investigated in 20 patients with an abnormality of the short arm of chromosome 12 (abn 12p) detected cytogenetically. Patients in the study had c/pre-B acute lymphoblastic leukemia (ALL) (nine children and three adults), T-ALL (three adults), acute myeloid leukemia (AML) (two adults), biphenotypic acute leukemia (Bip-L) (one adult), myelodysplasia (MDS) (one adult) and chronic myelomonocytic leukemia (CMML) (one child). Abnormalities of 12p comprised deleted (del)(12p) alone (seven cases), add(12p) alone (seven cases), del(12p) and add(12p) (one case) and balanced translocations of 12p to 1p13, 1q31, 10q11, 14q11 and 15q15 (one case of each). A novel, exon-specific RT-PCR assay identified breakpoints in ETV6 in nine of 19 cases, and showed breakpoints in intron 5 (seven cases of children with c-ALL), in intron 4 (in one adult with Bip-L) and in intron 2 (in one adult with AML). RT-PCR for the ETV6/AMLI fusion (tested in 19 cases) was positive using standard primers in five cases (four of which had shown rearrangements in intron 5) and occurred as a variant fusion in a sixth case (also positive for a rearrangement in intron 5) using 3' RACE PCR. Southern blotting confirmed rearrangements in intron 5 in the five cases available for analysis and revealed a rearrangement in intron 5 in one of 10 cases with no evidence of intron 5 involvement by RT-PCR. Rearrangements in intron 5 of ETV6 were found in eight of nine cases of children with c-ALL of which six carried the ETV6/AMLI fusion. Heterozygosity within intron 5 (revealed by the genomic probe B1) was found in seven of 11 cases tested. Deletion of one allele was indicated in three cases with del(12p) and one case with add(12p). This study, using a combination of ETV6 exon-specific RT-PCR, RT-PCR for ETV6/AMLI and Southern blotting has shown that rearrangement and/or deletion of ETV6 may occur in up to 70% of patients with abn 12p. Furthermore, 90% of children in this study with an abn 12p and c-ALL, carried a rearrangement of ETV6 in intron 5.