The PRDX4 gene located at Xp22 encodes for a member of the peroxiredoxin gene family. Genes within this family exhibit thioredoxin-dependent peroxidase activity and have been implicated in cellular functioning, including proliferation and differentiation. Recently, PRDX4 has been identified as a partner gene in a t(X;21) translocation in a patient with acute myeloid leukemia. To determine whether PRDX4 was involved in other translocations, leukemia cells from 15 patients with Xp22 abnormalities were screened for involvement of the gene using fluorescence in situ hybridization (FISH). One sample from a 41-year-old woman with acute lymphoblastic leukemia showed three signals when hybridized with the PRDX4 probe. Cytogenetic analysis of the sample had identified a t(X;18)(p22;q23). Assuming that the three signals indicated a break within the PRDX4 gene, we performed FISH experiments and successfully narrowed the breakpoint on chromosome 18 to a 50-kb region. Subsequent analysis using spectral karyotyping showed that the leukemic cells had undergone multiple rearrangements and that a third X chromosome was present, albeit rearranged. Additional FISH experiments revealed that the third PRDX4 signal was the result of a third copy of the gene. Analysis of the other rearrangements has helped to characterize the multiple abnormalities within the leukemic cells. The findings underscore the importance of using multiple techniques when analyzing complex chromosomal rearrangements in malignant cells.