Many proteins have been implicated in the physiological function of telomerase, but specific roles of telomerase-associated proteins other than telomerase reverse transcriptase (TERT) remain ambiguous. To gain a more comprehensive understanding of catalytically active enzyme composition, we performed affinity purification of epitope-tagged, endogenously assembled Tetrahymena telomerase. We identified and cloned genes encoding four telomerase proteins in addition to TERT. We demonstrate that both of the two new proteins characterized in detail, p65 and p45, have essential roles in the maintenance of telomere length as part of a ciliate telomerase holoenzyme. The p65 subunit contains an La motif characteristic of a family of direct RNA-binding proteins. We find that p65 in cell extract is associated specifically with telomerase RNA, and that genetic depletion of p65 reduces telomerase RNA accumulation in vivo. These findings demonstrate that telomerase holoenzyme proteins other than TERT play critical roles in RNP biogenesis and function.