The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by it's marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2;5)(p23;q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM) and anaplastic lymphoma kinase (ALK) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2;5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2;5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2;5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2;5)-positive clinical specimens and seven known t(2;5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2;5)-negative cases were negative by FISH, all eight t(2;5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2;5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2;5).