Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.