The release of cytochrome c from mitochondria results in the formation of an Apaf‐1–caspase‐9 apoptosome and induces the apoptotic protease cascade by activation of procaspase‐3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf‐1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf‐1 and thereby inhibits cytochrome c‐mediated activation of caspase‐9. Addition of purified Apaf‐1 to Hsp90‐depleted cytosolic extracts restores cytochrome c‐mediated activation of procaspase‐9. We also show that Hsp90 inhibits cytochrome c‐mediated oligomerization of Apaf‐1 and thereby activation of procaspase‐9. Furthermore, treatment of cells with diverse DNA‐damaging agents dissociates the Hsp90–Apaf‐1 complex and relieves the inhibition of procaspase‐9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c‐dependent apoptosis and for involvement of a chaperone in the caspase cascade.