Uterine leiomyomata are one of several benign tumors characterized by frequent chromosomal rearrangement involving 12q15. The 12q15 rearrangement in leiomyomata typically is manifested as t(12;14)(q15;q23-24), which has been hypothesized to create pathobiologically significant fusion transcripts derived from HMGA2 and RAD51L1. To explore further this hypothesis, we mapped chromosomal breakpoints in 38 uterine leiomyomata with rearrangements involving 12q15 using fluorescence in situ hybridization. Most tumors (n = 26) harbored der(14)t(12;14)(q15;q23-24), whereas chromosomes 1, 5, 8, and 10 were involved in rearrangements with 12q15 in six myomas. An additional six cases had more complex rearrangements, including breakpoints other than 12q15 or 14q23-24, inversions of chromosome 12, insertions of 12q15 into chromosome 14, or additional translocation partners. Breakpoints were mapped either 5' (centromeric) or 3' (telomeric) in the HMGA2 locus in 24 and nine cases, respectively; one tumor was a mosaic of cells with either 5' or 3' breakpoints. Breakpoints flanking the gene in both 5' and 3' regions were found in six cases. Analysis of one tumor by 3' rapid amplification of cDNA ends showed altered transcripts in which either exons 1-3 of HMGA2 were aberrantly spliced to cryptic sites in chromosome 12 or transcripts encompassing the full coding sequence of HMGA2 through a portion of the 3' untranslated region were fused to sequence from chromosome 14. A panel of 10 uterine leiomyomata with t(12;14) was specifically tested for fusion transcripts. RAD51L1-HMGA2 transcripts were not detected. HMGA2-RAD51L1 transcripts, however, were detected in four tumors; two of these tumors had uncommon rearrangements in the 3' region of HMGA2 and two had 5' rearrangements. Although the mechanism of fusion transcripts derived from tumors with 5' breakpoints is unclear, these findings indicate that formation of a fusion transcript is not the principle pathobiological mechanism in uterine leiomyomata. The pattern of rearrangements suggests dysregulated expression of HMGA2, most often by translocation of chromosome 14 sequence 5' to this gene.