Caspases (ICE/ Ced3 proteases) are a closely related family of cysteine proteases that play a key role in apoptotic cell death. We examined the role of caspases in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A. LLC-PK1 cells contain caspase activity that was markedly inhibited by cleavage site-based peptide inhibitors of caspases but not by inhibitors of serine, cysteine, aspartate or metalloproteinases. The caspase activity increased within five minutes of exposure to antimycin A, preceding any evidence of DNA damage and cell death. The specific caspase inhibitors. Ac-Tyr-Val-Ala-Asp-aldehyde (inhibitor I) and Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II) prevented, in a dose dependent manner, antimycin A-induced DNA strand breaks as determined by DNA unwinding assay (residual double stranded DNA in control, 94 +/- 2%; antimycin A alone, 48 +/- 3%; antimycin A + inhibitor I at 50 microM, 93 +/- 2%; antimycin A + inhibitor II at 50 microM, 89 +/- 5%; N = 3 to 4, P < 0.001). These inhibitors also prevented antimycin A-induced DNA fragmentation as determined by agarose gel electrophoresis and by in situ labeling of cell nuclei by the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) method. The caspase inhibitors markedly prevented antimycin A-induced cell death in a dose-dependent manner as measured by trypan blue exclusion (control 6 +/- 1%, antimycin A alone 40 +/- 1%, antimycin A + inhibitor I at 50 microM 16 +/- 1%, antimycin A + inhibitor II at 50 microM 16 +/- 1%; N = 4 to 7, P < 0.001). These data indicate that the caspase family of enzymes play an important role in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A.