A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).