We have previously shown that adipose cells from young genetically obese Zucker rats are characterized by very high metabolic activity together with an increase in a wide range of membrane-mediated functions. The aim of the present study was to examine whether the physical properties of the membranes and the composition of the membrane lipids were altered in these cells. Plasma membranes and two intracellular membrane fractions were prepared by differential ultracentrifugation from inguinal adipose cells of 30-day-old obese (fa/fa) and lean (Fa/fa) littermates. The lipid order as measured by steady-state fluorescence polarization of diphenylhexatriene used as probe was markedly decreased in the plasma membranes of obese rat adipose cells. Consistent with this, the cholesterol-to-phospholipid ratio was significantly decreased, and the degree of unsaturation of the phospholipid fatty acids was significantly increased. In intracellular membranes, none of these parameters were altered by the different genotype. In fat cells from obese rats, both plasma and intracellular membranes exhibited a 2-fold decrease in the ratios of n-6/n-3 fatty acids mainly due to an enrichment in docosahexaenoic acid (22:6n-3). The data show that the fatty genotype is a determinant of membrane lipid order and composition in adipose cells. The alterations reported here for young obese Zucker rat adipocytes might be related to the metabolic hyperactivity of these cells.