The non-esterified fatty acid (NEFA) content and phospholipid composition of mitochondria isolated from the livers of Wistar rats infected with Fasciola hepatica were examined in relation to the aberrant mitochondrial respiration previously reported [Rule, Behm, and Bygrave (1989) Biochem. J. 260, 517-523]. At 2 weeks post-infection, elevated NEFA levels were associated with uncoupling of mitochondrial respiration that was reversible in vitro by the addition of BSA. State IV respiration rates showed a strong correlation with NEFA content. At 3 weeks post-infection, NEFA content had increased further and uncoupled mitochondria no longer showed any response to BSA. 31P-NMR analyses of cholate extracts of mitochondria from infected livers at 3 weeks post-infection revealed a marked loss of several major phospholipid species with a concomitant increase in catabolic products, particularly glycerophosphocholine and glycerophosphoethanolamine. Similar changes were observed in microsomal extracts. The NEFA content and phospholipid composition of mitochondria isolated from infected, athymic nude rats were not significantly different from uninfected, athymic rats. These findings suggest that uncoupling of liver mitochondria during infection with F. hepatica is the result of phospholipase activation mediated by the immune system of the host.