When puparia of the onion maggot Delia antiqua were preexposed to 5°C for 5 days starting at different time points after pupariation, a large increase in survival after exposure to -20°C for 5 days was observed only when pre-exposure was initiated at 3-6 days after pupariation. The increase in cold hardiness was not associated with a large increase in the trehalose content of the puparia. The supercooling point of the puparia naturally decreased from -18 to -27°C in the first three days after pupariation, and pre-exposure to 5°C did not have an additional effect. Thus, factors responsible for the enhancement of cold hardiness by acclimation other than trehalose and supercooling point should be sought. The period of responsiveness to cold acclimation coincided with the time soon after head evagination, which corresponds to "pupation" in lepidopteran insects. The puparia appear to be physiologically flexible for a short time after head evagination, and able to adapt their physiology to the contemporary cold environment.