Butyrate, a short chain fatty acid produced in the colon, induces apoptosis in cancer cell lines by a sequential process involving inhibition of histone deacetylase, de novo protein synthesis and activation of DEVD-caspase, a major effector of apoptotic DNA fragmentation and membrane blebbing. We now show, in LIM 1215 colorectal cancer cells, that butyrate, in addition to activating DEVD-caspase and inducing apoptosis, also increases expression and cleavage of the universal cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and leads to hypo-phosphorylation of retinoblastoma protein. Accompanying these molecular changes was a progressive loss of G0/G1 and S phase cells. Expression of p21 had similar kinetics to that of the essential protein required for DEVD-caspase activation, indicating parallel effects of butyrate on anti-apoptotic and pro-apoptotic mechanisms, LIM 1215 cells, which were resistant to butyrate-induced apoptosis, were selected by three cycles of exposure to butyrate and removal of floating apoptotic cells. These cells showed markedly enhanced p21 expression and were in cell cycle arrest as determined by flow cytometry. On the other hand, subsequent culture of these cells for 2-3 days in the absence of butyrate resulted in down-regulation of p21 and restoration of sensitivity to apoptosis by butyrate. Western blots of butyrate-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band (p15) that was not present in control cultures. This band became apparent immediately after the onset of DEVD-caspase activation, was enriched in the floating apoptotic cell population when compared with the adherent, non-apoptotic cells and was absent in butyrate-resistant cells lacking DEVD-caspase activity. Peptide caspase inhibitors partially blocked appearance of p15, Here we show, for the first time, that p21 is a target of effector caspases in colorectal cancer cells and that the resistance to butyrate-induced apoptosis is characterized by failure of p21 cleavage.