The covalent coupling of a model steroid, 17 alpha-hydroxypregnenolone, to the wells of the microtiter plate, CovaLink NH, for use in a competitive enzyme-linked immunosorbent assay is described. This plate has secondary amino groups bound to its surface. A carboxylated derivative of the steroid was coupled to the amino group to form an amide bond in a single step using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (10 mM) as coupling reagent in the presence of N-hydroxysuccinimide (1 mM). After carrying out a competitive immune reaction, antibodies bound to immobilized steroids were estimated by means of a second antibody-enzyme conjugate. The non-specific background was reduced with blocking agents which did not interfere with the immune reaction between antibodies and the steroids coupled to the plastic surface. The following two procedures were effective for this purpose: pretreatment of wells with 0.01% Tween 20 solution followed by 0.5% bovine serum albumin in phosphate buffered saline, and addition of 0.01% Tween 20 to the assay buffer. With this method, the preparation of steroid-enzyme conjugates is unnecessary and optimization of conditions for ELISA procedures can be achieved in a simple manner.