Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosome 12 aberrations has indicated that their chromosome 12 breakpoints map between linkage locus D12S8 and the CHOP gene. Here, we report fluorescence in situ hybridization (FISH) and molecular cloning studies of the chromosome 12 breakpoints in a panel of seven such uterine leiomyoma cell lines; five with the frequently observed t(12;14)(q15;q24), one with t(12;15)(q15;q24), and one with ins(12;11)(q14;q21qter). Directional chromosome walking studies starting from D12S8 and the CHOP gene resulted in the isolation of a relatively large number of overlapping YAC clones, including Y5355 (465 kbp), Y7673 (360 kbp), and Y9899 (275 kbp). In total, the inserts of these three YAC clones span an 800 kbp long and presumably contiguous stretch of human genomic DNA. All chromosome 12 breakpoints of the uterine leiomyoma cell lines studied were found by FISH analysis to be mapping within a 445 kbp subfragment of this region and, furthermore, to be dispersed over a DNA region which is at least 150 kbp in size. The chromosome 12 breakpoint of t(12;14)(q15;q24) in uterine leiomyoma cell line LM-30.1/SV40 was tentatively mapped within the 60 kbp region between YAC clones Y9899 and Y5355. From this 60 kbp region and close to sequence-tagged site RM99, we isolated probe pRM118-A, which showed in Southern blot analysis that it detected a rearrangement in LM-30.1/SV40 DNA, and generated restriction maps of the normal and rearranged genomic DNA regions detected with this probe. Finally, we molecularly cloned part of one of those rearranged DNA fragments using a vectorette-PCR-based technique and demonstrated that it consisted of 12q13-q15 sequences fused to DNA sequences derived from 14q23-24 and most likely represented the translocation junction on der(14) in LM-30.1/SV40 cells. Our studies strongly suggest that we have identified and isolated the uterine leiomyoma cluster region of chromosome 12 breakpoints, which we designate ULCR12, and molecularly cloned and characterized the der(14) translocation junction in cells derived from a uterine leiomyoma carrying the frequently observed t(12;14)(q15;q24).