In the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation.