The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] dipalmitoyl-L--phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20°C) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20°C (13.2 ± 0.7°C)- than 5°C (7.2 ± 0.1°C)-acclimated trout. Similarly, acclimation from 5 to 20°C increased both the onset temperature (from 2.0 ± 0.3 to 7.2 ± 0.6°C) and the thermal range (from 10.9 ± 0.5 to 16.0 ± 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately 2°C) and completion temperatures (9°C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58°C; only membranes of 5°C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 ± 1.7°C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.