Aspirin and other NSAIDs reduce the incidence of and mortality from colon cancer, but their mechanism of action remains unknown. We evaluated the effect of aspirin (ASA) and three other structurally unrelated NSAIDs (indomethacin, naproxen, and piroxicam) on cell proliferation, cell cycle phase distribution, and the development of apoptosis in HT-29 colon adenocarcinoma cells in vitro. All of the NSAIDs examined reduced the proliferation and altered the morphology of these cells in a time- and concentration-dependent manner. In addition, they altered the cell cycle phase distribution of these cells. They increased the proportion of cells in the G0/G1 phase and reduced the proportion in the S phase of the cell cycle. ASA and indomethacin also reduced the percentage of cells in the G2/M phase, whereas naproxen and piroxicam did not. Parallel to their effect on cell cycle, ASA and indomethacin also reduced the levels of p34cdc2 and p33cdk2, two cyclin-dependent kinases that are important for cell cycle progression. Finally, all the NSAIDs analyzed, except ASA, induced apoptosis in these cells. There as a rough correlation between the relative potency of these compounds in inducing apoptosis and their effectiveness in retarding cell proliferation. Our findings indicate that NSAIDs can reduce the proliferation of HT-29 colon cancer cells in vitro. In addition, they cause cell cycle quiescence and apoptosis, both of which could account for their anti-proliferative effect. These findings suggest possible mechanisms for the cancer preventive effects of these compounds in humans.