Metaphase preparations from 36 patients with acute lymphoblastic leukemia (ALL) have been retrospectively screened by fluorescence in situ hybridization (FISH) to determine the incidence of translocation (12;21) and the potential usefulness of FISH as an adjunct to conventional cytogenetic analysis. With the use of specific chromosome paints, 4 of 31 patients with B-lineage childhood ALL (13%) demonstrated rearrangements of chromosomes 12 and 21, and therefore, were considered to harbor the translocation, which had not previously been detected by conventional karyotyping. However, none of these positive cases revealed the standard reciprocal t(12;21)(p12;q22) as the sole abnormality involving chromosomes 12 and 21. The study confirms the feasibility and advantages of introducing FISH screening for t(12;21) in pediatric ALL cases and demonstrates the usefulness of FISH screening as a backup to concurrent cytogenetic analysis to resolve variant translocations and aberrant results. The presence of t(12;21) has also been correlated to clinical data to assess the prognostic significance of this translocation on its own or in association with other prognostic features.