The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase-positive cells and alternative lengthening of telomeres (ALT) in telomerase-negative cells. We report here that telomere maintenance in the telomerase-positive human ovarian SKOV-3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase-positive cell line (i.e. human pharynx FaDu cells) and the telomerase-negative human osteosarcoma Saos-2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization-based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3'-azido-deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV-3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of approximately 80%) and by expression of antisense hTR (complete inhibition in SKOV-3 cells and maximal inhibition of approximately 80% in FaDu cells). However, treatment with telomerase inhibitors resulted in approximately 50% telomere shortening in FaDu cells but had no effect on SKOV-3 nor Saos-2 cells. SKOV-3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos-2 cells. Collectively, these results suggest the existence of a telomerase-independent mechanism of telomere maintenance in the telomerase-positive SKOV-3 cells.