The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype. The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation. Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established. By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement. The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.