Sex-steroid-binding plasma proteins (SBPs) of man, cattle, dog, and rabbit were purified to apparent homogeneity by sequential chromatography on testosterone-17 alpha-ethynylcarboxyaminoethyl Sepharose and hydroxyapatite. When subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, all the purified SBPs were resolved into two subunits, the relative amounts of which differed considerably from species to species. Two-dimensional electrophoresis according to O'Farrell also revealed that each subunit was further separable into several charged variants. The heavy subunit had somewhat more acidic molecular variants than the light subunit. One molecule of 5 alpha-dihydrotestosterone was bound per dimer of the subunits. Dissociation constants of heavy and light homodimers of rabbit SBP were 3.3 and 4.9 nM, respectively. Polypeptide fragmentation patterns resulting from digestion of heavy and light subunits with protease V8 differed from species to species but resembled each other in each species. These results suggest that the native SBPs may exist as a homodimer of a single variant or a hybrid dimer composed of various combinations of light and heavy variants.