Abstract: The telomerase ribonucleoprotein reverse transcriptase restores the simple sequence repeats at chromosome ends that are lost as a consequence of cellular proliferation. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble in a heterologous cell extract to form a catalytically active enzyme capable of reiteratively copying the TER-encoded template. However, the physiological process of telomerase biogenesis is more complex. Here, I reconstitute the hierarchical pathway of Tetrahymena telomerase biogenesis in vitro. Using a bacterially expressed polypeptide, I performed quantitative binding assays and defined TER sequence requirements for recognition by the N-terminal RNA interaction domain of TERT. This N-terminal domain and the high-affinity RNA binding domain of TERT have non-overlapping requirements for TER sequence and structure that together account for all of the sites of TER contact inferred for full-length TERT. While TER assembly with TERT must occur to form active enzyme, these two components alone will not stabilize each other in vivo; the endogenous accumulation of both requires an additional telomerase holoenzyme protein, p65. I demonstrate that four modules of p65 contribute affinity for the TER 3' stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, I show that recognition of a conserved GA bulge in TER by a novel C-terminal domain of p65 is both necessary and sufficient for this enhanced interaction of TERT with TER. Interestingly, I have identified regions of both TER and p65 that are not required for high affinity protein-RNA interaction but are required for increased assembly of TERT with p65-bound TER. These findings reveal a pathway of essential interactions for biogenesis of the telomerase holoenzyme.