The purpose of the study was to determine systematically the expression of telomerase activity and the length of telomere repeat arrays by utilizing two different cell culture models that derive from normal individual donors, and probably represent various stages of human breast oncogenesis in cell culture. The models consist of mortal, non-tumorigenic immortal and tumorigenic immortal human mammary epithelial cell (MEC) lines. Using a recently developed polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay, telomerase activity was undetectable in mortal MEC cells. In contrast, the immortal MEC that were nontumorigenic or tumorigenic in immunosuppressed athymic mice, showed telomerase activity. The absence of telomerase activity in mortal and its presence in both non-tumorigenic and tumorigenic immortal cell lines did not reflect their proliferative rate, as demonstrated by the similar pattern and intensity of reactivity of these cell lines with anti-Ki 67 antibody which recognizes a human nuclear cell proliferation--associated antigen. Southern blot analyses of Hinf I-digested genomic DNA hybridized with a (TTAGGG)4 probe revealed arrays of telomeric repeat lengths ranging from 3 to 5, 3.5 to 9, 3.2 to 9 or 3 to 15 kilobase pair (kbp) for mortal, nontumorigenic immortal, and tumorigenic immortal or established MEC lines respectively. These results suggest that telomerase activity and stable telomeric repeat lengths may be a molecular phenotype of the early stages in the progression of breast cancer.