There is experimental evidence that dietary fish oil, which contains the n-3 fatty acid family, i.e., EPA and DHA, protects against colon tumor development, in part by increasing apoptosis. Since mitochondria can act as central executioners of apoptosis, we hypothesized that EPA and DHA incorporation into colonocyte mitochondrial membranes, owing to their high degree of unsaturation, would enhance susceptibility to damage by reactive oxygen species (ROS) generated via oxidative phosphorylation. This, in turn, would compromise mitochondrial function, thereby initiating apoptosis. To test this hypothesis, colonic crypts were isolated from rats fed either fish oil, purified n-3 fatty acid ethyl esters, or corn oil (control). Dietary lipid source had no effect on colonic mitochondrial phospholipid class mole percentages, although incorporation of EPA and DHA was associated with a reduction in n-6 fatty acids known to enhance colon tumor development, i.e., linoleic acid LNA) and its metabolic product, arachidonic acid (ARA). Select compositional changes in major phospholipid pools were correlated to alterations in mitochondrial function as assessed by confocal microscopy. The mol% sum of LNA plus ARA in cardiolipin was inversely correlated with ROS (P = 0.024). Ethanolamine glycerophospholipid ARA (P = 0.046) and choline glycerophospholipid LNA (P = 0.033) levels were positively correlated to mitochondrial membrane potential. In contrast, ethanolamine glycerophospholipid EPA (P = 0.042) and DHA (P = 0.024) levels were negatively correlated to mitochondrial membrane potential. Additionally, EPA and DHA levels in choline glycerophospholipids (P = 0.026) were positively correlated with caspase 3 activity. These data provide evidence in vivo indicating that dietary EPA and DHA induce compositional changes in colonic mitochondrial membrane phospholipids that facilitate apoptosis.