1q rearrangement is a remarkably frequent secondary chromosomal change in both non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), where it is associated with tumor progression. To gain insight into 1q rearrangement-associated disease mechanisms, we used fluorescence in situ hybridization (FISH) to search for recurring 1q breaks in 35 lymphoma samples (31 NHL patients and 4 lymphoma-derived cell lines) as well as 22 MM patients with cytogenetically determined 1q abnormalities. Strikingly, dual-color FISH analysis with chromosome 1 centromere and 1q12-specific probes identified constitutive heterochromatin band 1q12 as the single most frequent breakpoint site in both NHL and MM (39% and 89% of 1q breaks, respectively). These rearrangements consistently generated aberrant heterochromatin/euchromatin junctions and gain of 1q12 material. A further 30% of NHL 1q breaks specifically involved two other novel, closely spaced sites (clusters I and II) within a 2.5 Mb region of proximal 1q21 (D1S3620 to D1S3623). A possible association between these sites and NHL subtype was evident; the cluster I rearrangement was frequent in follicular and diffuse large cell lymphoma, whereas the cluster II rearrangement was more frequently observed in diffuse small-cell lymphoma (2/2 marginal zone lymphomas, 1/2 atypical chronic lymphocytic leukemias, and 1 lymphoplasmacytic lymphoma in this series). Candidate oncogenes bordering this interval (BCL9 and AF1Q) were not rearranged in any patient except one (AF1Q). This study provides the first evidence of involvement of 1q12 constitutive heterochromatin in the pathogenesis of NHL and MM and indicates proximal 1q21 to be of specific pathological significance in NHL. © 2001 Wiley-Liss, Inc.