Studies have shown that immune responses are depressed in male mice, but not in proestrus females after trauma-hemorrhage (TH), resulting in increased mortality from subsequent sepsis in male mice compared with female mice. These gender-specific alterations in immune function are believed to be due to differences in sex steroid levels. Aromatase is a key enzyme in the sex steroid biosynthesis. Although earlier studies have shown that aromatase inhibitors prevent thymic atrophy in aged male rats, it remains unknown whether the use of 4-hydroxy-androstenedione (4-OHA) after TH in male mice has any salutary effects on the depressed immune responses. Male C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30+/-5 mmHg for 90 min) followed by adequate fluid resuscitation. 4-OHA (5 mg/kg) or vehicle was administrated s.c. just before resuscitation. At 2 h after resuscitation, the mice were killed, and spleens were harvested. Splenocyte proliferation, interleukin (IL-2), interferon (IFN-gamma), and IL-10 release and expression of androgen (AR) and estrogen receptors (ER)-alpha and -beta by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) were assessed. In another group, sepsis was induced by cecal ligation and puncture (CLP) 3 days after resuscitation, and survival was measured over a period of 10 days. A significant decrease in splenocyte proliferation, IL-2, and IFN-gamma release and increased release of IL-10 were observed in vehicle-treated mice. Animals treated with 4-OHA showed increased splenocyte proliferation, IL-2, and IFN-gamma release, and decreased IL-10 release. Immunoblot analysis showed decreased expression of the cytosolic AR, but no significant difference in the cytosolic and nuclear ER-alpha and -beta expression was observed in the vehicle-treated group after TH. In addition, AR and ER-beta mRNA expression was increased, whereas ER-alpha expression decreased in the vehicle-treated group after TH. ER-alpha expression decreased and ER-beta expression increased in the nucleus of 4-OHA treated mice as determined by immunoblot. There was no difference in the cytosolic AR expression in the 4-OHA-treated group after TH. AR and ER-beta mRNA expression was unaffected, whereas ER-alpha expression increased under such conditions. In additional groups, the increased mortality rate after TH and subsequent sepsis was significantly reduced by 4-OHA treatment. Thus, 4-OHA seems to be a novel and useful adjunct for restoring the depressed immune functions in males after TH and for decreasing mortality rates from subsequent sepsis.