A novel approach to the enzyme immunoassay of analytes is presented and illustrated by demonstration of an estradiol assay. The assay depends on the competitive binding of estradiol and estradiol-DNP conjugate to immobilized anti-estradiol. The amount of estradiol-DNP bound is inversely proportional to the amount of free estradiol, and is measured through the use of peroxidase-labeled anti-DNP antibodies. The assay gives a dose-response curve from 10 pg/tube to 450 pg/tube and shows satisfactory correlation with radioimmunoassay.