Adults of the insect Pyrrhocoris apterus acquire chill tolerance through the process of autumnal acclimatization. Field and laboratory experiments were conducted to separate the triggering effects of low temperatures, desiccation and diapause progression on the physiological characteristics related to chill tolerance with emphasis on the restructuring of glycerophospholipid (GPL) composition. Changes in relative proportions of major molecular species of glycerophosphoethanolamines (GPEtns) and glycerophosphocholines (GPChols) in thoracic muscle and fat body tissues were followed using HPLC coupled to electrospray ionisation mass spectrometry. The increase in relative proportion of 1-palmitoyl-2-linoleyl-sn-GPEtn at the expense of 1,2-dilinoleyl-sn-GPChol was the most prominent feature of the complex change observed in both tissues during autumnal acclimatization in the field. The relative proportion of total GPEtns increased, while the proportion of total GPChols decreased. The relative proportion of unsaturated fatty acyls slightly decreased. A similar restructuring response was seen during acclimatization in the field and cold acclimation in the laboratory. By contrast, the GPL changes related to desiccation and diapause progression were relatively small, differed qualitatively from the cold-acclimation response, and were accompanied with no increase of chill tolerance. Other features of autumnal acclimatization, i.e. depression of supercooling capacity and accumulation of polyhydric alcohols, were also triggered solely by low temperatures.