Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of and mortality from colon cancer. In addition, NSAIDs reduce the number and the size of polyps in patients with familial adenomatous polyposis. The mechanisms responsible for the antineoplastic effect of NSAIDs are not yet completely understood, but one of the possible mechanisms is an induction of apoptosis. We explored the role of caspase-3, a major apoptosis-executing enzyme, in NSAID-induced apoptosis of colon cancer cell line HT-29. Treatment of HT-29 cells with indomethacin induced a dramatic increase in caspase-3-like protease activity measured by a cleavage of the fluorogenic substrate Ac-DEVD-AMC. Western blot analysis showed that indomethacin treatment led both to decrease in procaspase-3 and to cleavage of its substrate poly(ADP-ribose) polymerase (PARP). Furthermore, the caspase-3-like protease inhibitor Ac-DEVD-CHO attenuated indomethacin-induced DNA fragmentation dose dependently. However, mRNA expression of CASP genes was not affected by the addition of indomethacin, highlighting the importance of posttranslational modification of this enzyme for the activation. These results suggest that NSAIDs, including indomethacin, induce apoptosis in colon cancer cells through a caspase-3 dependent mechanism which may contribute to the chemopreventive functions of these agents.