Telomerase is a ribonucleoprotein that participates in the maintenance of telomere length. Its activity is up-regulated in many tumor types, suggesting that it may be a novel target for chemotherapy. The RNA component of telomerase contains an active site that plays at least two roles&sbd;binding telomere ends and templating their replication [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337]. The accessibility of RNA nucleotides for inhibitor binding cannot be assumed because of the potential for RNA secondary structure and RNA-protein interactions. Here we use high-affinity recognition by overlapping peptide nucleic acids (PNAs) [Nielsen, P. E., et al. (1991) Science 254, 1497-1500] to identify nucleotides within the RNA active site of telomerase that are determinants for inhibitor recognition. The IC50 for inhibition decreases from 30 microM to 10 nM as cytidines 50-52 (C50-52) at the boundary between the alignment and elongation domains are recognized by PNAs overlapping from the 5' direction. As C50-52 are uncovered in the 3' direction, IC50 increases from 10 nM to 300 nM. As cytidine 56 at the extreme 3' end of the active site is uncovered, IC50 values increase from 0.5 microM to 10 microM. This analysis demonstrates that C50-C52 and C56 are important for PNA recognition and are physically accessible for inhibitor binding. We use identification of these key determinants to minimize the size of PNA inhibitors, and knowledge of these determinants should facilitate design of other small molecules capable of targeting telomerase. The striking differences in IC50 values for inhibition of telomerase activity by related PNAs emphasize the potential of PNAs to be sensitive probes for mapping complex nucleic acids. We also find that PNA hybridization is sensitive to nearest-neighbor interactions, and that consecutive guanine bases within a PNA strand increase binding to complementary DNA and RNA sequences.