A liquid phase "two-site" immunoradiometric assay (IRMA) specific for human thyroid stimulating hormone (hTSH) is described. The assay is based on the simultaneous addition of affinity purified sheep anti hTSH IgG-I 125 and rabbit anti hTSH antiserum to standards and unknowns followed by 4h incubation at room temperature. The separation of free labelled sheep IgG-I125 from that bound to hTSH is achieved by the addition of sheep anti-rabbit IgG Fc fragment antiserum. The radiolabelled sheep anti-hTSH IgG-I 125 was pretreated with solid phase urinary postmenopausal gonadotropins to remove cross reaction with FSH and LH. The assay is specific for hTSH and no cross reaction with the other anterior pituitary glycoproteins or protein hormones has been found. In addition it is characterized by a wide operating range, rapid equilibration of reactants and high sensitivity (0.02 microU/ml). The precision of dose estimates was less than 10% between 0.25-2.5 microU/ml and less than 2.5% over the range 2.5-60 microU/ml.