Telomerase is an unusual ribonucleoprotein that synthesizes new telomeres onto chromosome ends. The enzyme has been most extensively characterized in ciliates, where the RNA component has been cloned from several species, and its elongation properties have been characterized in detail. To understand the substrate specificity and protein composition of telomerase, we have used gel shift and UV cross-linking to characterize the enzyme from the ciliate Tetrahymena thermophila. In a mobility shift assay, a complex was identified that contained telomerase RNA, co-purified with telomerase activity, and was sensitive to nuclease treatment. The mobility shift complexes specifically formed using several different single-stranded, telomeric sequences but not non-telomeric primers. These results suggest that the specificity of telomerase for G-rich primer sequences occurs at least in part at the level of primer binding. UV cross-linking analysis identified a 100-kDa cross-linked protein that may be a telomerase component.