The ribonucleoprotein telomerase holoenzyme is minimally composed of a catalytic subunit, hTERT, and its associated template RNA component, hTR. We have previously found two additional components of the telomerase holoenzyme, the chaperones p23 and heat shock protein (hsp) 90, both of which are required for efficient telomerase assembly in vitro and in vivo. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR. We report here that the hsp70 chaperone also associates with hTERT in the absence of hTR and dissociates when telomerase is folded into its active state, similar to what occurs with other chaperone targets. Our data also indicate that hsp90 and p23 remain associated with functional telomerase complexes, which differs from other hsp90-folded enzymes that require only a transient hsp90⅐p23 binding. Our data suggest that components of the hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.Vertebrate telomeres are composed of the repeated sequence TTAGGG and are responsible for maintaining chromosomal stability and integrity (1). Conventional DNA polymerases are incapable of replicating to the end of a linear molecule (the end replication problem), resulting in loss of telomeric DNA during cellular proliferation of normal somatic cells (2, 3). The specialized reverse transcriptase, telomerase, compensates for this loss of telomere sequence and is responsible for maintenance and preservation of telomere ends in germ cells and immortal and cancer cells (4, 5). The reverse transcriptase subunit of telomerase, hTERT, contains the catalytic activity of the enzyme, whereas the associated RNA component, hTR, serves as the template for synthesis of telomeric sequences (6 -8). As direct evidence that telomere erosion plays a major role in cellular senescence, hTERT was ectopically expressed in normal human cells, which endogenously express hTR, resulting in activation of telomerase, stabilization of telomere lengths, and extension of cellular life span (9, 10).Expression of the hTERT and hTR components in heterologous systems has allowed for increased understanding of the biochemical features of the telomerase enzyme. Reconstitution of human telomerase activity has been accomplished in a variety of in vitro systems including yeast, baculovirus, rabbit reticulocyte, wheat germ, and human (6,7,11,12), and each has verified the essential role of hTERT and hTR in active telomerase. Recently, we have demonstrated that the hsp90 1 chaperone complex is required for assembly of human telomerase both in vitro and in vivo in a rabbit reticulocyte system and in human cells (13). We observe that hsp90 and p23 associate with hTERT in the absence of hTR and that the minimal components necessary for active telomerase assembly are hTERT, hTR, hsp90, p23, hsp70, p60, and hsp40/ydj. In addition, we demonstrate that geldanamycin, a p...