The plasma membrane lipid order of 3T3F442A cells was examined during the course of adipocyte differentiation by measuring the fluorescence polarization of 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene. This cationic fluorophore labels the plasma membrane but does not rapidly redistribute to intracellular organellar membranes and can, therefore, be used to specifically probe the plasma membrane of intact cells. Studies with whole cells demonstrated that the plasma membrane of 3T3F442A cells becomes less ordered during the course of adipocyte conversion and that this alteration begins relatively early during the differentiation process. In addition, the lipid order of plasma membranes isolated from adipocyte-stage cells was found to be lower than the lipid order of the early, fibroblast-stage cells. Analysis of membrane lipid composition suggests that the molecular bases for the decrease in adipocyte plasma membrane lipid order are a large increase in the level of monounsaturated phospholipid acyl chains and a decrease in the molar ratio of cholesterol to phospholipid. The alteration in plasma membrane lipid composition may be specifically required for integral membrane protein function, since the differentiation-dependent fatty acid desaturase activity is known to be maintained even in the absence of triacylglycerol accumulation.