Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether L-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (cNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the "stretch PCR" method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four L-enantiomers of cNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition modes of L-dTTP and L-dGTP mere partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K-i values of L-dTTP and L-dGTP (21 and 15 muM) were several times larger than the K-m values (3-6 muM). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT.