The steroid hormone receptors in N-2-fluorenylacetamide (2-FAA)- and N-hydroxy-2-FAA-induced mammary adenocarcinomas in outbred Sprague-Dawley rats were analyzed. Both 8S and 4S estrogen-binding components have been detected in cytosols of these tumors following sucrose gradient sedimentation in low salt. Competitive binding analyses of this binder indicated a specificity profile expected of an estrogen receptor. Both androgen and progesterone receptors were also present in the cytosols of these mammary tumors. While the androgen receptor sedimented in the 8S region of the gradient, the progestin binder appeared only as a 4S moiety under similar conditions. The relative concentrations of these receptors (expressed in fmol/mg protein +/- SE) were: 17 beta-estradiol (28.6 +/- 4.1) greater than 5 alpha-dihydrotestosterone (8.5 +/- 2.2) greater than progesterone (5.0 +/- 1.3). The progesterone receptor was increased at least eightfold in the mammary adenocarcinomas from ovariectomized rats that were treated with diethylstilbestrol for 6 days. Binding equilibrium data indicated Ka = 1.2-1.8 X 10(9) M-1 for the above cytoplasmic hormone receptor complexes (Ka, association constant). Although cytosols prepared from lactating mammary gland contained appreciable quantities of glucocorticoid receptor, only trace amounts were found in the mammary adenocarcinoma.