Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of <sup>125</sup>I to give a specific activity of 0.041 µCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing <sup>125</sup>I-prolactin from antibody. High specific activity <sup>125</sup>I-prolactin (1 iodine atom/molecule; 70 µCi/µg) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro <sup>125</sup>I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.