The hydrodynamic properties of rat hepatic prolactin receptors have been determined by a combination of gel chromatography and ultracentrifugation. Prolactin receptors were detergent extracted from partially purified plasma membranes prepared from female rat livers. Fifteen different nonionic detergents were tested for solubilizing prolactin receptors, including Triton X-100, Polyoxyethylene W-1, Lubrol WX, detergents of the Tween and Brij series, and digitonin. When the receptors were detergent solubilized after ligand was bound to the receptor, 1% Triton X-100 had the highest efficacy of solubilization. However, if the receptors were solubilized prior to exposure to ligand, maximum binding was to receptors solubilized with 0.25% Triton X-100. The Kd of 43.2-74.5 pM for binding to the soluble receptor was three- to fivefold lower than the Kd for the membrane receptor. Gel chromatography (Bio-Gel A-1.5m, 2.5 x 50 cm) of the soluble receptor indicated a Stokes radius (Rs) of 5.0 nm for the hormone-receptor-detergent complex. The hydrodynamic properties of the receptor-detergent-ligand complex were determined by centrifugation in 5-20% sucrose gradients in H2O and in D2O. They are v = 0.7; S20,w = 4.7; f/f0 = 1.49; Mr = 118,000 for the complex, 73,000 for the receptor alone. Approximately 0.22 mg of Triton X-100 is estimated bound per milligram of protein. This represents about 25 mol detergent/mol receptor.