Cytosols from 75 normal, 36 abnormal, and 5 decidual human endometrial tissue specimens were assayed for the presence of a high affinity, progesterone-specific binding protein. Thirty of the normal and 15 of the abnormal samples were found to contain a binder which would form a high-affinity complex with progesterone but not with cortisol, 17beta-estradiol, testosterone, or 5alpha-androstane-3-,17-dione. Incubation of cytosol with trypsin or incubation for 2 hours at 37 C abolished [3H]progesterone binding by these preparations, indicating the protein nature and heat-lability of the binder. The average equilibrium constant of dissociation, Kd, of the progesterone-binder complex was 4.0 X 10(-10)M in each phase of the menstrual cycle. The concentration of the binder varied over the cycle, however, with a significant peak at mid-cycle (P = .02). The average saturation values in femtomoles (fmoles)/mg protein ranged from 21 in the early proliferative phase to 64 in the late proliferative samples, dropping to 36 in early secretory and to 3 in the late secretory phase of the cycle. No progesterone-specific binding was detected in decidual samples. Saturable binding was demonstrable in 10 of 22 endometrial hyperplasias, 80-1840 fmoles/mg protein, with high affinity, Kd 3.3 X 10(-10)M. Two other hyperplasia samples bound progesterone, but with lower affinity. Two grade I adenocarcinomas, one grade III adenosquamous carcinoma, and one grade III adenocarcinoma contained the progesterone binder, but in 9 other cancers no detectable binder was present. A benigh adenocanthomyoma was found to contain a progesterone binder (18 fmoles/mg protein with a Kd of 2.5 X 10(-10)M).