The reason for estrogen independence of C3H mouse mammary tumors has been sought in the initial steps of estradiol action. The characteristics of the estrogen receptors were similar to those observed in estrogen-responsive tissues: high affinity and binding specificity, DNA binding and 8S sedimentation constant as shown by sucrose gradient centrifugation. Their concentration averaged 18.5 +/- 3.5 (S.E.) fmol/mg cytosol protein in the cytosol and 3.5 +/- 1.0 fmol/mg cytosol protein in the KCl nuclear extract. The nuclear translocation of the cytosol receptor was investigated with the use of biopsy and in vivo injections of radioactive estradiol. No nuclear translocation of estrogen receptor could be ascertained with the dextran-coated charcoal assay since the free and nonspecifically bound estrogen conjugate(s) were also assayed by this technique. However, when the estrogen-receptor complexes were estimated by more specific methods such as protamine sulfate or hydroxylapatite precipitations, the estrogen receptor translocation into the nucleus was clearly shown. We therefore conclude that the estrogen independence of C3H mammary tumors cannot be explained by a defect in the two initial steps of the mechanism of action of estradiol, namely, cytosol binding and nuclear translocation of receptors.