At low temperature, cell membrane functionality depends on adjustments of membrane phospholipid fatty acid (PLFA) composition. We here test the hypothesis that previous exposure to copper (Cu) may deteriorate tolerance to freezing temperatures because of Cu-induced changes of PLFA composition of cell membranes in the freeze-tolerant earthworm Dendrobaena octaedra. Cu levels and freezing temperatures were varied in a full factorial design. We measured PLFA composition and lipid peroxidation. A highly significant interaction was observed between subzero temperatures and Cu concentrations above 120 mg/kg dry soil. Lipid peroxidation increased slightly in worms exposed to Cu. In particular, the analysis showed that Cu had a significant negative effect on the polyunsaturated PLFA, linoleic acid (18:2 omega 6,9), which has previously been reported to correlate positively (R-2 = 0.92) with freeze tolerance in D. octaedra. This supports our hypothesis that reduced tolerance to freezing temperatures in Cu-exposed worms may be due to membrane damage.