Abstract Human pregnancy serum was previously reported to exhibit a high binding affinity for testosterone; steroid binding was measured by a semimicrotechnique, based on the principle of equilibrium dialysis but utilizing Sephadex G-25 in a batchwise fashion. This property may be ascribed to a testosterone-binding component, presumably a protein, present in very low concentration in serum. The testosterone-binding component was readily separated from corticosteroid-binding globulin (both steroid-binding proteins are present at elevated levels in late pregnancy) by column chromatography of pooled pregnancy serum on microgranular diethylaminoethyl cellulose. Stepwise elution furnished four major fractions: γ-globulin, β-globulin, albumin, and α-globulin, in that sequence. The testosterone-binding component appeared in the β-globulin fraction, whereas the corticosteroid-binding globulin (measured by its cortisol- and progesterone-binding activities) appeared in the albumin fraction; the latter fraction also exhibited considerable testosterone-binding activity, attributable to albumin itself and, in part, to corticosteroid-binding globulin. A portion of the β-globulin fraction (after precipitation in 50% ammonium sulfate) was chromatographed on a column of Sephadex G-100; the testosterone-binding protein was thereby separated from the bulk of the protein (the former emerged last from the column). Considerable losses in testosterone-binding activity were, however, encountered at each stage of purification, and so the over-all increase in specific binding activity (i.e. testosterone-binding activity per g of total protein) was only about 4-fold. Other fractionation procedures, i.e. column chromatography on hydroxylapatite and on fibrous DEAE-cellulose, were not as efficacious, but nonetheless informative; the testosterone-binding component is readily precipitated from whole serum in 50% saturated ammonium sulfate. The testosterone-binding component is distinctly different from corticosteroid-binding globulin in its chromatographic behavior and steroid-binding properties. There is also an appreciable difference in molecular size according to gel filtration studies: the testosterone-binding component has a Stokes molecular radius of about 47 A compared to 36 A for corticosteroid-binding globulin; the same value was obtained for the latter protein from the sedimentation and diffusion data of other investigators.