The hamster ductus deferens cloned tumor cell line (DDT1) contains a complex steroid receptor protein that binds 3H-labeled 5 alpha-dihydrotestosterone,. Diethylaminoethyl (DEAE) chromatography of cytosol from these cells yields two major receptor peaks of activity. Identification of this steroid binding protein as a cytoplasmic receptor was confirmed by salt dissociation on sucrose gradients, stability of the hormone-receptor complex at 0 degrees C, and the retention patterns on phosphocellulose and DEAE cellulose. Multiple forms of the receptor exist in a single homogeneous cell type. The data support the theory that steroid hormones bind to a cytoplasmic protein receptor composed of dissimilar subunits as the initial step in steroid hormone action.