The purpose of this work is to develop the procedures for the preparation of the reagents suitable for the radioreceptor assay (RRA) of human prolactin (Prl). Human purified Prl (NIAMDD-hPrl-16) was labelled with 125I by the Chloramine-T or alternatively by the lactoperoxidase method. As reference preparation we used Prl isolated from the ethanolic step of the routine procedure for the preparation of human growth hormone (hGH) for clinical purposes. The lactogenic receptors were prepared from the pregnant rabbit mammary gland previously stimulated with insulin, cortisone and dried thyroid extract. The final receptor preparations obtained by ultracentrifugation contained 8.85-39.36 mg protein per ml. The prolactin was measured in the human sera and in our hPrl preparations by a double antibody radioimmunoassay (RIA) system using the NIAMDD reagents. We developed a RRA system for hPrl using rabbit mammary receptor preparations with a protein concentration of about 4 mg/ml. The comparative competition showed the same magnitude of the inhibition of the tracer receptor binding of hPrl and hGH. This interference of hGH makes difficult the assessment of the specificity of the hPrl-RRA system otherwise accountable by the structural and/or biological relationship of the three lactogenic hormones: hPrl, hGH and human placental lactogen (hPL). Studies concerning the preparation of a purified and solubilized rabbit mammary receptor and of an antiserum for it are in progress in our laboratory with the objective to provide a useful tool for the investigation of the lactogenic receptor structure and function relationship.