This study examined endotoxin-mediated cytokinemia during exertional heat stress (EHS). Subjects were divided into trained (TR, n=12, VO_2peak = 70 ±2 mL·kgLBM^-1·min^-1) and untrained (UT, n=11,VO_2peak = 50 ± 1 mL·kgLBM^-1·min^-1) groups prior to walking at 4.5 km·h^-1 with 2% elevation in a climatic chamber (40°C; 30% R.H.), wearing protective clothing until exhaustion (Exh). Venous blood samples at baseline and 0.5°C rectal temperature increments (38.0°C, 38.5°C, 39.0°C, 39.5°C and 40.0°C/Exh) were analyzed for endotoxin, lipopolysaccharide binding protein (LBP), circulating cytokines and intranuclear NF-B translocation. Baseline and Exh samples were also stimulated with LPS (100 ng·mL^-1) and cultured in vitro in a 37°C water-bath for 30 min. Phenotypic determination of natural killer (NK) cell frequency was also determined. Enhanced blood (104 ± 6 vs. 84 ± 3 mL·kg^-1) and plasma (64 ± 4 versus 51 ± 2 mL·kg^-1) volumes were observed in TR compared to UT. EHS produced an increased concentration of circulating endotoxin in both TR (8 ± 2 pg·mL^-1) and UT (15 ± 3 pg·mL^-1) (range ND to 32 pg·mL^-1) corresponding with NF-B translocation and cytokine increases in both groups. In addition, circulating levels of TNF- and IL-6 were also elevated combined with concomitant increases in IL-1ra in both groups and IL-10 in TR only. Findings suggest that the threshold for endotoxin leakage and inflammatory activation during EHS occurs at a lower temperature in UT compared to TR, and supports the endotoxin translocation hypothesis of exertional heat stroke, linking endotoxin tolerance and heat tolerance.