Previously, we developed methodology for studying allelic imbalance (AI) in preinvasive breast disease and showed that AI identified at various chromosomal loci in invasive carcinoma is already present in in situ carcinoma and atypical hyperplasia. We now extend this work by looking for Ai in hyperplasia of usual type (HUT), apocrine cysts (AC), and papilloma (Pap) of the breast. HUT, AC, and Pap were identified in formalin-fixed, paraffin-embedded sections of benign breast biopsies and isolated using a microdissection technique. AI was investigated using polymorphic microsatellite markers and PCR. AI was identified in 3/23 (13%) informative cases of HUT at 17q (D17S250), 2/43 (4.7%) at 17p (D17S796), 1/22 (4.5%) at 16q (D16S413), and 0/18 (0%) at 13q (D13S267). No particular histologic feature of HUT predicted the presence of AI. No examples of AC or Pap exhibited AI at any of the markers studied. AI previously identified at various chromosomal loci in invasive carcinoma, in situ carcinoma, and atypical hyperplasia is present at low frequency in HUT in benign breast biopsies but not in AC or Pap. The possibility that AI in the latter could be masked by contamination from stromal and myoepithelial cells cannot, however, be excluded at this stage. At least a proportion of HUT thus appear to be clonal (neoplastic) rather than hyperplastic proliferations as their name suggests. The significance of AI in the pathogenesis of HUT or its subsequent progression to carcinoma is not yet clear and requires further investigation.