A highly specific, high affinity binding protein for estradiol-17beta (E2) is present in cytosol prepared from the epididymides of immature (21-53 day old) rabbits. This binding moiety sediments on sucrose gradients as an 8S species under low ionic strength conditions and as a 4S species under conditions of high ionic strength (0.3 M KCL). The relative binding affinities of estrogens for the binding protein was E2 is greater than estrone is greater than estriol. Neither 5alpha-dihydrostestosterone (5alphaKHT), progesterone, nor cortisol were able to inhibit binding of [3H]E2 to epididymal binding sites. An 8S binding moiety for E2 was present in testicular cytosol but not in muscle. An apparently non-specific binding component for E2 was present in plasma which sedimented in the 4S region of low ionic strength gradients. The epididymal E2 binding moiety was distinct from a 4S androen binding protein of testicular origin which is detectable in cytosol prepared from epididymides of rabbits at certain stages development. We were unalbe to detect a specific E2 binding protein in epididymal cytosol from mature intact or 4-day castrated rabbits. The E2 binding component in the cytosol of immature rabbits had an Kd congruent to 2-10 X 10-10 M and the concentration of binding sites was in the order of 1-4 X 10-13 mmoles/mg of protein. The binding component was thermo-labele and pronase, but not nuclease, sensitive.