Abstract Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) are freeze tolerant in winter. During freezing, cell membranes must compensate for both low temperature and partial dehydration. Documented adaptations to low temperature include increased fatty acid unsaturation and enrichment of cone-shaped phosphatides, both of which inhibit formation of gel phase lipid domains. These changes appear inconsistent with adaptations known to prevent formation of the hexagonal II phospholipid phase at low water activities, namely, increased fatty acid saturation and increased proportions of cylindrical phosphatides. To address these inconsistencies, changes in E. solidaginis phospholipid composition and class-specific fatty acid composition were studied from August to November 2002. Cylindrical phosphatides, mostly phosphatidylcholine (PC), increased transiently and significantly, from 35% of the total to nearly 50%, during the transition from freeze susceptible to freeze tolerant. Monoenes in both PC and phosphatidylethanolamine (PE) represented 35% of total fatty acids in freeze-susceptible larvae but accumulated in PC to 48% and in PE to 42% in freeze-tolerant larvae. Moreover, PC accumulated the most unsaturated acid in this species, 18:3(n-3), to a significantly greater degree than PE. This combination of changes may represent a finely tailored response to both low temperatures and freeze-induced dehydration.