Fluorescent probes located in different membrane regions were used to evaluate effects of ethanol (50 and 100 mM) on structural parameters (protein distribution, fluidity of total and annular lipid, and thickness of the bilayer) of synaptic plasma membranes (SPMs) from brain cortex of High‐Alcohol Sensitivity (HAS) and Low‐Alcohol Sensitivity (LAS) rats. An experimental procedure based on radiationless energy transfer from tryptophan of membrane proteins to pyrene, 1,3‐bis‐(1‐pyrene)propane(pyr‐C3‐pyr), or 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), as well as pyr‐C3‐pyr monomer‐eximer formation and DPH polarization, and energy transfer from pyrene monomers to 1‐anilinonaphthalene‐8‐sulfonic acid (ANSA) was utilized. The efficiency of energy transfer from tryptophan to pyrene was sensitive to protein clustering induced in SPMs by concanavalin A. Efficiency of energy transfer from pyrene monomers to ANSA was different for vesicles of dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, and distearoyl phosphatidyl choline, consistent with differences in the thickness of these lipid bilayers. Without ethanol, there were no significant differences between the structural parameters of SPMs from HAS and from LAS rats. Addition of ethanol (50 mM) changed protein distribution (increased clustering) only in membranes from HAS rats and had no effect on the structure of membranes from LAS rats. A larger concentration of ethanol (100 mM) changed the fluidity of annular and total lipid in both lines of rats, but changed protein distribution and decreased thickness of the membranes from HAS rats with no effect on these parameters in SPMs from LAS animals. Ethanol (50 and 100 mM) decreased the binding affinity of ANSA for membranes from HAS, but not LAS rats. Genetic differences in ethanol sensitivity of HAS and LAS rats may be related to effects of ethanol on protein mobility, protein‐protein interactions, or membrane thickness, but do not appear to be due to effects of ethanol o