An assay for the cytoplasmic estrogen receptor in calf, human and rat uterus has been developed. The method is based on partial separation of free and bound estradiol (E2) by means of an aqueous two-phase system containing dextran and poly(ethylene glycol), respectively, in the two phases. Low-speed supernatant from uterus homogenate is equilibrated with E2 and [3H]E2. A two-phase mixture is then added and bound E2 will partition into the lower phase while free E2 is distributed in both phases according to its partition coefficient. The amounts of bound and free E2 are calculated and the receptor concentration and association constant are obtained from a Scatchard plot. No dissociation of bound E2 in the phase system could be demonstrated at 4 degrees C. The interassay coefficient of variation for receptor concentration at 4 degrees C was 20 and 14% for calf and human uterus, respectively. The intraassay variation for receptor concentration in calf uterus determined at 4 degrees C and 23 degrees C was 7.1 and 4.1%, respectively. The influence of freezing the tissue and supernatant preparation was examined and results from supernatant preparations obtained with different centrifugations were compared. The method is simple and rapid, permitting large numbers of samples to be handled efficiently by a single technician.