The molecular pathology of myotonic dystrophy is believed to be expressed at the plasma membrane level. Previous assessments of membrane fluidity, a marker of the biochemical state of the membrane, have yielded conflicting results. In this study, erythrocyte membrane fluidity was reevaluated using highly sensitive fluorescence probe techniques. Steady-state anisotropy was measured with diphenylhexatriene (DPH), trimethylaminophenyl-hexatriene (TMA-DPH) and phenylhexatrienylphenylpropionic acid, probing different regions of the membrane. In the patients, significantly increased steady-state anisotropy was obtained with DPH, probing the hydrophobic core of the membrane, while slightly reduced anisotropy was found with TMA-DPH. The dynamic properties of the membrane lipids were further examined by means of time-resolved measurements with DPH. The excited state decay kinetics could best be described by a bi-exponential decay model. A large redistribution of the probe populations and a reduction of the average order parameter were found in the patients indicating a less ordered or more fluid lipid matrix. These perturbations might be induced by a protein abnormality and altered protein-lipid interaction within the erythrocyte membrane.