Interphase fluorescence in situ hybridization (iFISH) was used independently to reveal chromosomal abnormalities of prognostic importance in a large, consecutive series of children (n = 2367) with acute lymphoblastic leukaemia (ALL). The fusions, TEL/AML1 and BCR/ABL, and rearrangements of the MLL gene occurred at frequencies of 22% (n = 447/2027) (25% in B-lineage ALL), 2% (n = 43/2027) and 2% (n = 47/2016) respectively. There was considerable variation in iFISH signal patterns both between and within patient samples. The TEL/AML1 probe showed the highest incidence of variation (59%, n = 524/884), which included 38 (2%) patients with clustered, multiple copies of AML1. We were thus able to define amplification of AML1 as a new recurrent abnormality in ALL, associated with a poor prognosis. Amplification involving the ABL gene, a rare recurrent abnormality confined to T ALL patients, was identified for the first time. The use of centromeric probes revealed significant hidden high hyperdiploidy of 33% and 59%, respectively, in patients with normal (n = 21/64) or failed (n = 32/54) cytogenetic results. The iFISH contributed significantly to the high success rate of 91% (n = 2114/2323) and the remarkable abnormality detection rate of 89% (n = 1879/2114). This study highlights the importance of iFISH as a complementary tool to cytogenetics in routine screening for significant chromosomal abnormalities in ALL.