To examine the telomere maintenance mechanism in the process of human fibroblast immortalization, we studied telomere length, telomerase activity, chromosome instability, and minisatellite alterations in human fibroblasts following the introduction of the human papillomavirus type 16 E6 gene or E7 gene, or both. One cell line immortalized by E6 alone maintained short telomeres, and its telomerase activity was positive. Fairly long and heterogeneous telomeres were maintained in all four E7-immortalized cell lines lacking telomerase activity. Of the three clones immortalized by both E6 and E7, two cell lines with telomerase activity maintained short telomeres, and the other cell line, which lacked telomerase activity, maintained long and heterogeneous telomeres. Although all immortal cell lines expressed mRNAs for human telomerase RNA component and telomerase-associated protein, expression of mRNA for human telomerase reverse transcriptase was detected only in the telomerase-positive cell lines. All immortal cell lines showed both chromosomal abnormalities, including structural and numerical changes, and minisatellite alterations detected by DNA fingerprinting. These findings indicate the existence of different telomere maintenance mechanisms in telomerase-positive and -negative fibroblast cell lines immortalized by E6, E7, or E6/E7, and the possible involvement of chromosome instability and minisatellite alterations in the activation of the telomere maintenance mechanisms.