The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential^1–3. Telomerase is an enzyme that restores telomeric DNA sequences⁴, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo⁵. Telomerase activity has been detected in 50–100% of tumors of different types, but not in most normal adult somatic tissues^6,7. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date⁷. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization^8–11 presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres)¹¹. Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.