A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, N-ethylmaleimide. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations. These 'non-reacting' SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labeled N-ethylmaleimide or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with N-[ethyl-2-3H]ethylmaleimide. The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive. After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards N-ethylmaleimide. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.