Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, causes disease much more frequently in men than it does in women, suggesting that the hormonal milieu of the host might influence P. brasiliensis pathogenicity. We recently demonstrated that cytosol from yeast cultures of P. brasiliensis contains a high-affinity, low-capacity, tritiated 17 beta-estradiol [( 3H]estradiol)-binding protein. Estradiol and, to a lesser degree, diethylstilbestrol (DES), inhibited the transformation of P. brasiliensis cultures from the mycelial to the yeast form, an event critical to the establishment of infection. Our current studies demonstrated a somewhat higher affinity (apparent dissociation constant [Kd], approximately equal to 6 to 12 nM) of the estrogen-binding protein for [3H]estradiol than was previously described for yeast cytosol. The presence of both high- and low-affinity estrogen-binding sites in yeast-form P. brasiliensis cytosol was detected after warming the cytosol to 37 degrees C. The high-affinity protein was labile to further heating (56 degrees C), although the low-affinity protein was stable. Additional experiments demonstrated the presence of an estrogen-binding protein in cytosol prepared from mycelial-form P. brasiliensis. This estrogen-binding protein had a slightly lower affinity for [3H]estradiol (Kd approximately equal to 13 nM), and its cytosol contained somewhat fewer binding sites (approximately equal to 78 fmol/mg of protein) than did yeast-form P. brasiliensis cytosol. Of particular interest was the finding that DES, a weak competitor for [3H]estradiol binding in yeast cytosol, displaced [3H]estradiol from the mycelial-form binding moiety. DES had a 50- to 100-fold-lower affinity for the [3H]estradiol-binding protein than did estradiol, consistent with its lower bioactivity in the mycelial-to-yeast-form transformation studies. The current results lend further support to our hypothesis that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast-form transformation, thus explaining the resistance of women to paracoccidioidomycosis.