Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonasexotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFalpha. Also the fluorescent substrate, DEVD-AFC, is cleaved 2-4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than untreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38-induced PARP cleavage is inhibited by several protease inhibitors known to inhibit caspases (zVAD-fmk, zDEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additional evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and loss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill cells. Despite inhibition of apoptosis, the immunotoxin is still capable of selective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the arrest of protein synthesis caused by inactivation of elongation factor 2. The fact that an immunotoxin can specifically kill tumor cells without the need of inducing apoptosis makes such agents especially valuable for the treatment of cancers that are protected against apoptosis, e.g., by overexpression of Bcl-2.