Although 17β-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)-α or ER-β and which signaling pathways are involved in such 17β-estradiol effects. Utilizing ER-α- or ER-β-specific agonists, this study examined the role of ER-α and ER-β in 17β-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-κB are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER-α agonist propyl pyrazole triol (PPT; 5 μg/kg), the ER-β agonist diarylpropionitrile (DPN; 5 μg/kg), 17β-estradiol (50 μg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF-α production and activation of MAPK and NF-κB were measured. Macrophage IL-6 and TNF-α production and MAPK activation were decreased, whereas NF-κB activity was increased, following trauma-hemorrhage. PPT or 17β-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17β-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER-α plays the predominant role in mediating the salutary effects of 17β-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-κB signaling pathways. Copyright © 2007 the American Physiological Society.