Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60 v-src . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7⌬ slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7⌬ growth defect. Overexpression of CNS1 in cpr7⌬ cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.Cells rely on molecular chaperones to facilitate the folding of nascent polypeptides, prevent protein aggregation, and enable proper inter-and intramolecular interactions. Indispensable in both Saccharomyces cerevisiae (5) and Drosophila melanogaster (13), Hsp90 is one of the most abundant and highly conserved chaperones. In vitro, Hsp90 displays general chaperone properties by preventing the aggregation of proteins such as citrate synthase (52) and casein kinase II (30) and by maintaining -galactosidase (21) in a folding-competent state. In vivo, however, Hsp90 is not required for the folding of most proteins (33) but instead plays a key role in the maturation of a small subset of proteins typically involved in signal transduction (3,32,42). It is needed for the maturation of certain steroid hormone receptors (40,43,44), basic helix-loop-helix transcription factors (45