The availability of [125I]-16α-iodo-3,17β-estradiol ([125I]-E2) with binding characteristics similar to estrogen receptor (ER) enabled us to establish a double-labeling assay for the simultaneous determination of ER and progesterone receptors (PgR) using 125I-E2 and [3H]-R5020. The criteria for the establishment of such a double-labeling assay are described. 150 human mammary tumor cytosols have been investigated with the standard routine receptor assay for ER as well as PgR and the results were compared to those obtained by the double-labeling assay. ER: a coefficient of correlation of 0.691 was obtained, the parameters of the regression line were Y = 1.025 x X-0.036. When referring to the standard assay, 3 determinations were false-positive and 4 false-negative. PgR: a correlation coefficient of 0.984 was found, the parameters of the regression line were Y = 0.960 × X + 12.16. One value was false-negative and 1 false-positive. An equivalence of the two methods could be demonstrated. This new assay reduces by half the amount of tissue necessary for a valid 4-to 6-point saturation analysis, the time required for performing the assay and its co