The present study was conducted to assess whether Premarin, a water-soluble estrogen sulfate, can act via estrogen receptors (ERs) to rescue mice from heat-induced lethality. Unanesthetized, unrestrained mice were exposed to ambient temperature of 42.4°C to induce heatstroke (HS). Another group of mice was exposed to room temperature (24°C) and used as normothermic controls. They were given isotonic sodium chloride solution, Premarin (0.1 - 1.0 mg/kg of body weight, i.p.), or Premarin (1 mg/kg of body weight, i.p.) plus the nonselective ER antagonist ICI 182, 780 (0.25 mg/kg of body weight, i.p.) 1 h after the termination of heat stress. Their physiologic and biochemical parameters were continuously monitored. Mice that survived on day 4 of heat treatment were considered survivors. When the vehicle-treated mice underwent heat, the fraction survival and core temperature at +4 h of body heating were found to be 0 of 12 and 34.4°C ± 3°C, respectively. Administration of Premarin (1 mg/kg) 1 h after the cessation of heat stress rescued the mice from heat-induced death (fraction survival, 12/12) and reduced the hypothermia (core temperature, 37.3°C). The beneficial effects of Premarin in ameliorating lethality and hypothermia can be abolished by simultaneous administration of ICI 182, 780. Both IL-10 (an anti-inflammatory cytokine) and estradiol in the serum were increased significantly in heat-stressed mice administered Premarin compared with vehicle-treated HS group. Heat-induced apoptosis, as indicated by terminal deoxynucleotidyl-transferase-mediated αUDP-biotin nick end-labeling staining, in the spleen, liver, and kidney were significantly reduced by Premarin. The increased levels of cellular ischemia (e.g., glutamate, lactate-to-pyruvate ratio, and nitrite) and damage (e.g., glycerol) markers and iNOS expression in the hypothalamus during HS were decreased significantly by Premarin therapy. The levels of proinflammatory cytokines (e.g., IL-1β and TNF-α) and renal and hepatic dysfunction markers in plasma that are up-regulated in heat stressed mice were significantly lower in Premarin-administered mice. The data indicate that Premarin may act via ERs to rescue mice form HS-induced lethality.