Three distinct groups of mitogen-activated protein kinases (MAPKs) have been identified in mammalian cells (i.e., ERK, JNK, and p38) which play an important role in the differentiation and apoptosis of various cells. The purpose of our present study was to determine MAPK activity and levels associated with sodium butyrate (NaBT)-mediated differentiation and apoptosis in the human colon cancer cell lines Caco-2 and HT29. Intestinal alkaline phosphatase (IAP) activity, a marker of intestinal differentiation, was increased at 48 h after NaBT treatment followed by cell death at 72 h. ERK activity was decreased in differentiated Caco-2 cells either induced with NaBT or allowed to differentiate spontaneously and in HT29 cells treated with NaBT. The combination of the MEK inhibitor, PD98059, with NaBT further increased IAP activity and cell death compared with NaBT alone. In contrast to ERK, JNK1 activity and c-Jun phosphorylation was increased 8 h after NaBT treatment suggesting a role for the JNK pathway in intestinal cell differentiation and apoptosis. p38 activity was increased at 24 and 48 h after NaBT treatment. Taken together, our results suggest that alterations in MAPKs (i.e., ERK inhibition and JNK induction) contribute to the differentiation and apoptotic pathways in intestinal cells.