Butyrate is a potentially selective therapeutic agent for many adenocarcinomas. Butyrate causes reversible growth arrest as well as some death of VACO 5 colon cancer cells. Combined treatment with butyrate and the phorbol ester TPA leads instead only to cell death, while TPA causes little death on its own. Cells dying during treatment with TPA and butyrate, as well as those dying in the presence of butyrate alone, exhibit features typical of apoptosis, including detachment, shrinkage and internucleosomal DNA cleavage. Pre-treating VACO 5 cell cultures with TPA for as little as 6 hr prior to butyrate addition led to a markedly diminished enhancement of butyrate-induced apoptosis. Treatment with a distinct PKC activator, bryostatin 1, was ineffective in enhancing butyrate-induced death and, furthermore, counteracted the death-enhancing actions of TPA. Such antagonism was apparent when bryostatin was added after 12 hr of TPA/butyrate treatment but was much less effective thereafter. The duration of TPA/butyrate treatment required for depressing cell survival by >95% was thereby estimated to be 24 hr. Other colon cancer cell lines were examined for the extent of cell death following treatment with TPA/butyrate. In each of these lines, butyrate inhibited cell replication in a reversible manner, similar to that seen in VACO 5. However, the combination of butyrate and TPA led to high levels of cell death in only a subset of these lines. TPA/butyrate-treated cultures of COLO 201 exhibited extensive apoptosis, similar in timing and magnitude to the response by VACO 5, whereas HCT 116 was reversibly growth-arrested. Our findings indicate that the PKC system plays a critical role in maintaining cell survival during butyrate-induced growth arrest.