Crystallization temperature (T(c)), resistance to inoculative freezing (IF), ice contents, bound water, protein and glycogen body contents were measured in the aquatic subterranean crustacean Niphargus rhenorhodanensis and in the morphologically close surface-dwelling aquatic crustacean Gammarus fossarum, both acclimated at 12 degrees C, 3 degrees C and -2 degrees C. Cold acclimation induced an increase in the T(c) values in both species but no survival was observed after thawing. However, after inoculation at high sub-zero temperatures, cold-acclimated N. rhenorhodanensis survived whereas all others, including the 3 degrees C and -2 degrees C acclimated G. fossarum died. In its aquatic environment, N. rhenorhodanensis is likely to encounter inoculative freezing before reaching the T(c) and IF tolerance appears as a highly adaptive trait in this species. Bound water and glycogen were found to increase in the 3 degrees C and -2 degrees C acclimated N. rhenorhodanensis, whereas no variation was observed in G. fossarum. Considering the hydrophilic properties of glycogen, such a rise may be correlated with the increased bound water measured in cold-acclimated N. rhenorhodanensis, and may be linked to the survival of this species when it was inoculated. The ecological significance of the survival of the aquatic subterranean crustacean to inoculative freezing is paradoxical, as temperature is currently highly buffered in its habitat. However, we assume that past geographical distribution and resulting life history traits of N. rhenorhodanensis are key parameters in the current cold-hardiness of the species.