The isolation of brush-border membranes from trout enterocytes is described for both middle and posterior intestine. Both procedures are based on differential centrifugations combined with calcium precipitation. Classical marker enzymes are quantified and indicate a valuable purification of the membranes (13-18-fold). No difference appears when comparing the relative amounts of phospholipids, cholesterol and proteins in microvillus membranes isolated from either middle or posterior intestine. In contrast, the membranes isolated from middle intestine are more unsaturated than those from the posterior one, and their sphingomyelin/phosphatidylcholine ratio is lower. These differences are reflected by fluorescence anisotropy studies with diphenylhexatriene as lipid fluorophore which indicate a higher fluidity of the microvillus membranes from the middle intestine as compared with those from the posterior intestine. These results point out the importance of the fatty acyl chains and that of the relative amounts of phosphatidylcholine and sphingomyelin in controlling the fluidity of biological membranes in relation with their transport properties.