Membrane dielectric as a primary basis for effects of ethanol was examined in synaptic plasma membranes (SPM) of genetically selected ethanol-sensitive long-sleep (LS) and ethanol-resistant short-sleep (SS) mice. Multifrequency phase and modulation fluorometry of diphenylhexatriene (DPH) was used to resolve structural and dielectric differences in the membrane interior core. Fluorescence spectral peak ratios, fluorescence lifetime analysis, and initial rates of photoreaction of DPH in SPM provided sensitive measures of SPM interior core dielectric properties. The membrane microenvironment sensed by DPH was more polar in SPM from SS mice than in SPM from LS mice. Physiological concentrations of ethanol in vitro (25-75 mM) increased the SPM interior core dielectric and potentiated photoreaction of DPH with other membrane components of SPM from LS, but not SS, mice. These effects of ethanol in vitro were maximal by 75 mM ethanol and/or exacerbated at higher ethanol. In addition, ethanol in vitro increased the fraction of DPH associated with photoreaction products with lipids from SPM of ethanol-sensitive LS mice. The data were consistent with ethanol in vitro increasing the polar molecules (ethanol and/or water) of SPM from LS but not SS mice. It is suggested that ethanol alters the polarity and increases reactivity of the interior core lipid-protein interface.