The motional properties of phospholipids in bovine and rat liver microsomes and aqueous dispersions of the extracted lipids have been investigated employing 31 P NMR techniques. 2. The 31P NMR spectra obtained from the microsomes indicate that a considerable portion of the constituent phospholipids experience isotropic motion on the NMR timescale (10(-5) s). This is in strong contrast to the spectra obtained from aqueous dispersions of the extracted lipids, which display the characteristic lineshape associated with liquid crystalline phospholipids in (large) bilayer structures, which experience restricted anisotropic motion. 3. Evidence is presented which strongly suggests that the isotropic motion of microsomal phospholipids does not arise from tumbling of the microsomal vesicles or from lateral diffusion of phospholipids around these vesicles. 4. These results are discussed in terms of possible transitory formation of intramembrane non-bilayer lipid configurations, with which the bulk (bilayer) phospholipids are in rapid exchange.