Dimethyl sulfoxide (DMSO) is an organic solvent with myriad biological actions, including actions on synaptic membrane transport processes. In this study, fluorescence polarizations of the probes diphenylhexatriene (DPH: a probe of the hydrophobic membrane core), trimethylammonium-diphenylhexatriene (a probe of the superficial domain of the cytofacial synaptic membrane leaflet) and diphenylhexatriene propionic acid (a probe of the superficial domain of the exofacial synaptic plasma membrane leaflet) were measured in isolated rat cerebral synaptic plasma membranes. DMSO, added in vitro, increased fluorescence polarization of all of these intramembranous probes, an effect opposite that observed with the addition of ethanol. The fluorescence polarization increase appeared at lower concentrations of DMSO for the superficial membrane region probes (6% vol/vol DMSO) than for the membrane core probe (10% vol/vol DMSO). This is again in contrast to the effects of ethanol, which required lesser concentrations to decrease fluorescence polarization of DPH (50 mM ethanol) than that of the derivative probes (200 mM ethanol). The enhancement of DPH fluorescence polarization produced by DMSO was antagonized by the concomitant addition of ethanol. These results suggest an ordering effect of DMSO on synaptic plasma membranes, with greater effects in superficial membrane domains.