Upon treatment with various anticancer drugs, myeloid leukemia U937 cells undergo apoptosis. In this study, we found that either etoposide (VP-16) or camptothecin (CPT) activated c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK), transient c-jun expression, and ICE (interleukin-1beta converting enzyme)/CED-3-like proteases in U937 cells. Phorbol ester-resistant U937 variant, UT16 cells, displayed a decreased susceptibility to apoptosis induced by these drugs. The drugs did not cause JNK1 activation, c-jun expression, nor activation of ICE/CED-3-like proteases in UT16 cells. As reported previously, benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp), a preferential inhibitor of ICE/CED-3-like proteases, blocked the apoptosis of U937 cells. Interestingly, however, Z-Asp did not inhibit JNK1 activation in either VP-16- or CPT-treated U937 cells. The JNK1 antisense oligonucleotides diminished protein expression of JNK1 and inhibited drug-induced apoptosis of U937 cells, whereas sense control oligonucleotides did not. Consistent with this observation, the antisense oligonucleotide-treated cells did not respond to VP-16 or CPT with Z-Asp-sensitive proteases. These results indicate that JNK1 triggers the DNA damaging drug-induced apoptosis of U937 cells by activating Z-Asp-sensitive ICE/CED-3-like proteases.