Studies have shown that 17β-estradiol has salutary effects on immune functions after trauma-hemorrhage (TH). It remains unknown, however, whether 17β-estradiol has a similar effect in a double-hit model of TH and subsequent sepsis. It is also unknown if under those conditions the circulating immune cells accurately represent immunological responses occurring in fixed tissues, such as the spleen. To study this, pre-castrated mice were hormonally treated and then subjected to soft-tissue trauma (i.e. midline laporatomy), hemorrhagic shock (MAP 35±5mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMφ) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5α-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMφ and PBMC. In contrast, the 17β-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17β-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17β-estradiol were limited to tissue-fixed Mφ, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population. © 2003 Elsevier Ltd. All rights reserved.