The administration of reference endotoxin (Escherichia coli O:113, Lot EC-5) to humans has been an important means to study inflammation in vivo; however, the supply of Lot EC-5 is depleted. A new lot of reference endotoxin (Clinical Center reference endotoxin [CCRE]), derived from the original bulk material extracted from E. coli O:113, was processed. The effects of 0-, 1-, 2-, and 4-ng/kg doses of intravenous CCRE and EC-5 were studied in 20 male subjects. CCRE resulted in dose-related increases in symptoms, temperature (P = .016), total leukocyte count (P = .014), tumor necrosis factor—α (P = .004), interleukin (IL)—1 receptor antagonist (P = .004), IL-6 (P = .005), IL-8 (P = .011), cortisol(P < .05), and C-reactive protein (P = .04). These responses were attenuated (all P < .012) in subjects given Lot EC-5 (4 ng/kg) in comparison with those in subjects given CCRE, showing that, over several years, EC-5 had lost potency. Thus, in healthy subjects, the magnitude of exposure to CCRE results in a graded dose response of major components of innate immunity.