A two-step-method is described by which human and canine prealbumin have been isolated from serum in good yield and high purity. The first step involves binding and release of prealbumin by a thiol-disulfide interchange reaction with thiol-Sepharose 4B. The second step consists of reversible binding of prealbumin to retinol-binding protein covalently linked to Sepharose 4B. 2. The canine prealbumin was immunochemically identified as an alpha2-globulin. Like human prealbumin it is a tetramer with some 10% smaller subunits than human prealbumin. Both proteins are devoid of carbohydrate and both bind thyroxin.