To validate a 2-step FISH assay for the identification of the t(9;11)(p22;q23), 96 acute myeloid leukemias were studied by cytogenetic analysis, FISH and molecular biology. After a first FISH step using an MLL probe, 24/27 cases with 11q23 break showed MLL rearrangement. Southern blotting confirmed FISH data. In the second step, 24 cases with MLL rearrangement were studied using MLL and AF9 probes: 17/18 cases with t(9;11) showed MLL/AF9 fusion. In 6 patients with 11q23/MLL rearrangements other than t(9;11), FISH confirmed MLL involvement and excluded AF9 involvement. This is a reliable method for the identification of MLL/AF9 fusion in interphase cells, allowing for a reclassification of cases with suboptimal chromosome morphology. The frequency of deletion surrounding MLL and AF9 breakpoint is low.