A comprehensive understanding of the cellular functions of the Hsp90 molecular chaperone has remained elusive. Although Hsp90 is essential, highly abundant under normal conditions, and further induced by environmental stress, only a limited number of Hsp90 "clients" have been identified. To define Hsp90 function, a panel of genome-wide chemical-genetic screens in Saccharomyces cerevisiae were combined with bioinformatic analyses. This approach identified several unanticipated functions of Hsp90 under normal conditions and in response to stress. Under normal growth conditions, Hsp90 plays a major role in various aspects of the secretory pathway and cellular transport; during environmental stress, Hsp90 is required for the cell cycle, meiosis, and cytokinesis. Importantly, biochemical and cell biological analyses validated several of these Hsp90-dependent functions, highlighting the potential of our integrated global approach to uncover chaperone functions in the cell.