The receptor-like nature of previously-observed specific 4S estrogen binding in adult virgin female mouse mammary tissue cytosol has been further substantiated. The binding component does not arise from a plasma protein contaminant, nor is it an α-fetoprotein-type binder as evidenced by lack of affinity for diethylstilbestrol. The 4S protein binds 17β-estradiol with very high affinity (KA = 3 × 1010M−1), transforms to a 5.5 S form which translocates to nuclear binding sites under appropriate in vitro conditions. In vivo cytosol-nuclear binding distribution results are analogous to other estrogen receptor systems. During pregnancy, the 4S receptor is gradually replaced by an 8S form. This 8S complex does not represent a concentration-dependent aggregation of 4S subunits, as does the 8S receptor encountered in aged mice. The change in receptor concentration throughout pregnancy and lactation reflects the overall increase in tissue weight, and no major changes occur in specific binding activity, expressed on the basis of DNA content, cytosol protein concentration or unit weight of tissue. A single injection of 0.2 mg of prolactin to an adult virgin mouse results in an acute stimulation of receptor number, and a shift in molecular form from 4S to 8S. The effect is more pronounced if 3 successive daily injections of prolactin are administered, and thyroidectomy enhances the sensitivity of the receptor system to prolactin stimulation. Although the equilibrium binding constants are similar for the 4S and 8S complexes, analysis of the kinetics of formation and dissociation revealed that the 8S complex forms less readily, but has a half-life equal to 3 times that of the 4S complex. The differences in the amount, molecular nature and reactivity of the mammary tissue estrogen receptors, and the influence of specific hormonal elements on these parameters, set the stage for analysis of cellular response and responsiveness under diverse physiological and pathological conditions.