Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms characterized by frequent chromosome arm 14q losses. In this study, the 14q changes in a series of 39 histologically and immunohistochemically confirmed GISTs were analyzed in detail by metaphase and/or interphase fluorescence in situ hybridization (FISH) studies using 21 genetically well-characterized, region-specific 14q11-24 YAC clones. By conventional cytogenetic analysis, acquired clonal chromosome aberrations were found in 17 out of 35 tumors. Chromosome 14 was involved in 13 cases; six specimens showed complete chromosome 14 loss, while the remaining seven had structural abnormalities with the breakpoints residing within the intervals 14q11-13 or 14q22-24. Other recurrent chromosome aberrations included frequent deletions of chromosome 1p (11/17), losses of chromosome 22 (7/17), losses or deletions of chromosome arm 13 (6/17) or 15 (4/17), and gains or translocations involving chromosome 17 (4/17). Combining cytogenetic data with double-color FISH analysis, total or partial losses of 14q material were detected in 29 out of 36 tumors (81%). The 14q losses were found in all stages and histological subtypes. Two most frequent common deletion regions flanked by YACs 931B1 and 761D4, and 802E7 and 892C11 at 14q23-24 (25/30 of each; 83%) could be identified. Furthermore, 21 tumors (70%) shared a region of deletion defined by YACs 957H10 and 931E5 at 14q11-12. Our results suggest the presence of at least three distinct critical deletion regions on chromosome 14 in GISTs.