We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide‐induced cytotoxicity by preventing apoptosis. As observed for Bcl‐2, HSP27 overexpression delays poly(ADP‐ribose)polymerase cleavage and procaspase‐3 activation. In contrast with Bcl‐2, HSP27 overexpression does not prevent etoposide‐induced cytochrome c release from the mitochondria. In a cell‐free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase‐3 in both control and bcl‐2‐transfected U937 cells but fails to activate procaspase‐3 in HSP27‐overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP‐mediated activation of procaspase‐3. Overexpression of HSP27 also prevents procaspase‐9 activation. In the cell‐free system, immunodepletion of HSP27 increases LEDH‐AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide‐induced apoptosis by preventing cytochrome c and dATP‐triggered activity of caspase‐9, downstream of cytochrome c release.—Garrido, C., Bruey, J.‐M., Fromentin, A., Hammann, A., Arrigo, A. P., Solary, E. HSP27 inhibits cytochrome c‐dependent activation of procaspase‐9. FASEB J. 13, 2061–2070 (1999)