Detection of allelic loss in human breast cancer is hindered by the fact that breast cancer tissues are frequently infiltrated by stromal and inflammatory cells. For this study, we carefully microdissected infiltrating breast cancer tumor cells from contaminating normal cells and analyzed the DNA from these samples for allelic loss on the long arm of chromosome 6 by using a panel of 15 dinucleotide repeat markers. We found 53 of the 66 cases studied (80%) to have allelic loss of either the entire chromosomal arm (37 cases) or a portion of the chromosomal arm (16 cases). One common region that was identified for all tumors with deletions of 6q was the area between markers D6S310/314 and D6S473/255, consistent with a tumor suppressor gene locus at 6q23-6q25.2. The use of tissue microdissection allowed the detection of allelic loss in this chromosomal region in human breast cancer at a much higher frequency than was previously recognized.