The role of cholesterol in the thermal adaptation of biological membranes is explored. Physical and chemical responses of membranes to acclimation temperature were evaluated using plasma membrane domains (basolateral and brush border) prepared from intestinal epithelia of 5- and 20 °C-acclimated rainbow trout (Oncorhynchus mykiss). Basolateral membranes (BLMs) exhibit perfect homeoviscous efficacy (indicated by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene), although cholesterol content does not change with acclimation temperature (molar ratios of cholesterol to phospholipid are 0.23± 0.01 from 5 °C-acclimated fish and 0.25±0.02 from 20°C-acclimated fish; mean ± s.e.m.). Reductions (greater than 30 %) in each of the two major saturated fatty acids (16:0 and 18:0), and a 42 % increase in the polyunsaturate 22:6 (n-3) are found in BLMs from fish acclimated to 5 °C compared with membranes from warm-acclimated animals, suggesting that the phospholipid acyl chain composition determines the physical properties of BLMs. In marked contrast, brush-border membranes (BBMs) display opposite trends. BBMs from 5 °C-acclimated fish are more ordered than BBMs from 20 °C-acclimated fish (inverse compensation). Cholesterol content expressed relative to protein or relative to total polar lipid (phospholipid plus glycolipid) is significantly higher in cold- than in warm-acclimated fish, and nearly so (P=0.15) relative to phospholipid (0.31±0.03 in 5 °C-acclimated animals and 0.25±0.02 in 20 °C-acclimated animals). Only minor changes in the acyl composition of BBMs are induced by temperature acclimation. These results suggest that bile, a constituent of the apical microenvironment, may impose unusual requirements for membrane order and/or stability in the brush border.