We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.