To test the feasibility of using fluorescent in situ hybridization (FISH) on interphase and metaphase cells to detect numerical aberrations in childhood acute lymphoblastic leukemia (ALL), we analyzed bone marrow of 15 patients with cytogenetically documented hyperdiploidy with more than 50 chromosomes at diagnosis. Patients were selected on the basis of being trisomic or tetrasomic for chromosomes 17 and/or 18 as determined by G-banded chromosome analysis. We performed a double target FISH using DNA probes specific for the centromeric region of chromosomes 17 and 18, respectively. The numerical changes regarding chromosome 17 and/or 18 identified by FISH on metaphases were found in all cases analyzed by FISH on interphase nuclei. In 8 of 15 patients, FISH on interphase nuclei demonstrated the presence of one or more groups of cells with different combinations of trisomy and tetrasomy of the two chromosomes investigated, beside the ones detected on metaphases. Overall our findings indicate that interphase FISH analysis could be a useful method to detect the presence of numerical aberrations of two chromosomes simultaneously in bone marrow and peripheral blood specimens of ALL as an adjunct to conventional cytogenetic investigation or metaphase FISH.