Tritiated promegestone ([3H]R5020) is bound with high affinity in cytosol prepared from the thymus gland of both male and female rats. To prevent tracer binding to glucocorticoid receptors, we have used excess RU26988 (11 beta, 17 beta-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-trien-20-yn-3-one), a highly specific synthetic glucocorticoid. In the presence of 1 microM RU26988, [3H]R5020 was bound with high affinity (Kd less than or equal to 1 nM) and specificity (R5020 greater than progesterone much greater than estradiol greater than dihydrotestosterone = cortisol = corticosterone = dexamethasone) appropriate for progesterone receptors (PR). These sites were predominantly located in the epithelioid fraction of the thymus. PR levels were 14 +/- 3 fmol/mg protein in female thymus and 10 +/- 4 fmol/mg in male thymus; estrogen administration elevated thymic PR levels in both species (female, 26 +/- 2 fmol/mg; male, 21 +/- 3 fmol/mg). In estrogen-primed female rats, cytosolic thymic PR levels were acutely lowered to 20% of estrogen-primed control levels by the im administration of 1 mg progesterone. Thymic PR levels in pregnant (11-14 days) rats were approximately 50% of those in nonpregnant controls. Effects of glucocorticoids, androgens, and estrogens on the thymus are well documented; the demonstration of PR raises the possibility of direct effects of progesterone on the thymus.