Individuals of the freeze-tolerant earthworm, Dendrobaena octaedra, and four freeze-intolerant earthworm species (Dendrodrilus rubidus, Aporrectodea icterica, A. caliginosa, and A. longa) were frozen at -2 degree C. Control earthworms were exposed to +2 degree C. 1H nuclear magnetic resonance spectroscopy-based metabolic profiling in combination with multivariate pattern recognition methods (metabonomics) was used to produce a cross-species comparison. Several biochemical changes were detected as a result of freezing in all worm species, including an increase in relative free alanine concentrations, and an apparent conversion of adenosine to inosine. It was also possible to determine a number of biochemical changes that were unique to the freeze-tolerant species, D. octaedra. The most obvious difference was that, although all species showed an increase in glucose concentrations, the increase was largest in D. octaedra, and was coupled with a concomitant decrease in glycogen. This confirms that--like previously studied freeze-tolerant earthworm species--tolerance is effected by rapid glucose production from glycogen reserves. An additional difference noted was that succinate increased in all species on freezing, but the increase was least in D. octaedra. Furthermore there was no lactate accumulation in D. octaedra, whereas three of the other four species accumulated lactate. This indicates that anoxic metabolism was lowest in the freeze-tolerant species.