Estrogens have multiple influences on immune functions. Estrogen receptors (ERs) have two distinct subtypes - alpha and beta. To explore the specific roles of each ER subtype in estrogen-mediated immunomodulation, we investigated the effects of ER subtype-selective agonists on immune functions in ovariectomized Balb/c mice. Treatment with ERalpha-selective agonist propyl pyrazole triol (PPT) caused thymic atrophy and significant changes in thymic CD4/CD8 phenotypic profile. In contrast, ERbeta-selective agonist diarylpropionitrile (DPN) alone had no effect on thymic weight, cellularity or CD4/CD8 phenotype expression. When coadministered with PPT, DPN partially antagonized PPT-evoked decrease in thymic cellularity and also partially attenuated PPT-induced shifts in thymic T-cell phenotype. These results indicate that ERalpha plays a predominant role in estrogen-induced thymic atrophy and ERbeta activation may partially down-regulate ERalpha-mediated effects on thymic cellularity and T-cell phenotype expression. In addition, PPT administration induced a reduction in the percentage of mature B cells in the spleen, and enhanced IFN-gamma production but suppressed IL-6 production from in vitro Con A-stimulated splenocytes as estradiol (E(2)) did, whereas DPN treatment had no effects either alone or with PPT, suggesting ERalpha mediates these estrogen actions. Treatment with PPT or DPN did not augment anti-DNP antibody production after DNP-KLH immunization as E(2) did, implying that not merely one ER signaling pathway is involved in mediating estrogen's effects on specific humoral immune responses. Our study further indicates ER subtype-selective agonists provide a novel approach to explore each ER subtype-mediated immunomodulation.