Abstract Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human α 2-macroglobulin (α2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au 1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the α2M-Nanogold conjugates (α2M-Au 1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed α2M molecules. From this study we propose three possible locations for the cysteine 949.