The comparative affinity of steroidal and non-steroidal estrogens for binding sites in rabbit uterine cytosol was determined quantitatively using an in vitro assay system. It was found that with few exceptions relative binding affinity paralleled uterotropic activity. Binding affinity of steroids depended strongly on the phenolic hydroxyl and on substitutions in ring D. Additional substitutions of oxygen functions or unsaturation of the estra-triene nucleus were inhibitory. Synthetic estrogens competed and synthetic progestogens did not compete for cytosol binding sites. This binding assay may be used to determine estrogenic potency, though it must be borne in mind that binding does not distinguish between estrogens and competitive inhibitors.