The samples comprised normal tissues from 10 cases with carcinoma and from 3 reduction mammoplasties. Two cases had a family history of breast disease, one with a known germline mutation in the BRCA1 gene. A piece of fresh tumour tissue with some adjacent normal tissue was used for the in vitrocloning. For cloning, the tissues were disagregated with collagenase and cultured on mouse 3T6 fibroblasts. After 10-14 days the clones consisted of 1000-5000 cells and were identified as either of luminal or myoepithelial origin using phase-contrast morphology. The clones were then individually harvested by conventional ring-cloning techniques.For the LOH analysis tumour and normal duct-lobular units were dissected using the fine point of a glass pippette. LOH was performed by amplification of polymorphic microsatellite markers using fluorescence tagged primers and the polymerase chain reaction. The dinucleotide repeats used were D3s1597 (3p24.2), D3s1578 (3p21.1), D11s902 (11p15), D13s267 (13q13), D16s413 (16q24.3), D17s796 (17p13.2) and D17s250 (17q12). A value of 0.5 or less was used as indicative of LOH.Comparative genomic hybridisation (CGH) analysis was carried out on one case that showed LOH at the same allele within an invasive tumour and one luminal and one myoepithelial clone. Sequence analysis in the patient with a known BRCA1 mutation was also performed.