Telomerase is a specialized reverse transcriptase, which catalyzes the addition of telomeric repeats to the 3' ends of linear chromosomes using its integral RNA subunit as the template. An active Tetrahymena thermophila telomerase complex can be reconstituted in vitro from two essential components, tTERT, the catalytic protein subunit, and tTR, the RNA subunit. While the sequence specificity of telomerase has been investigated using template sequence mutants, there is no information regarding its backbone specificity. To address this question, we engineered two mutant forms of the telomerase RNA subunit that contain DNA only in the templating region and used rabbit reticulocyte lysates to reconstitute telomerase activity with the chimeric tTRs. The resultant telomerase mutants were able to extend telomeric DNA primers, albeit with reduced efficiency compared to the wild type. The reduced activity is presumed to be a function of the nascent DNA-template duplex structure. Additionally, the DNA-dependent telomerase mutants were RNase-sensitive, confirming that nontemplate portions of tTR are critical for maintaining activity of the telomerase ribonucleoprotein complex even after it is assembled. The splint ligation approach that we outline will allow the generation of tTR mutants containing a variety of nucleotide analogues, facilitating more elaborate studies of the interactions between the telomerase template and active site.