LMO4, a member of the LIM-only family of zinc-finger proteins, is overexpressed in more than 50% of primary breast cancers and cell lines, implying a role in the pathogenesis of this cancer. Southern blot analysis of these cell lines did not reveal amplification or rearrangement of the LMO4 gene. To investigate further the mechanism underlying LMO4 overexpression and the generation of two patterns of transcripts, we isolated genomic clones spanning the human gene. Similar to the mouse Lmo4 gene, there are two 5' noncoding exons, exon 1a and exon 1b, which we show are differentially expressed in breast epithelial cells. This reflects differential promoter usage in combination with alternative splicing. Two promoter regions were defined, one upstream of exon 1a and the other upstream of exon 1b. Both promoters exhibited strong activity in breast cancer cells, with up to 400-fold activity above basal levels. These promoters were significantly more active in T-47D and MCF-7 cells relative to SKBR3 cells, consistent with RNA levels. Thus, overexpression of the LMO4 gene in breast cancer cells reflects increased promoter activity and appears to involve aberrant activation of the second promoter in a subset of these cells.