The female sex hormone 17 beta -estradiol (E-2) has been shown to increase lipid and decrease carbohydrate utilization in animals. We administrated oral E-2 and placebo (randomized, double blind, crossover) to eight human male subjects for 8 days (similar to3 mg/day) and measured respiratory variables, plasma substrates, hormones (E-2, testosterone, leptin, cortisol, insulin, and catecholamines), and substrate utilization during 90 min of endurance exercise. [6,6-H-2]glucose and [1,1,2,3,3-H-2]glycerol tracers were used to calculate substrate flux. E-2 administration increased serum E-2 (0.22 to 2.44 nmol/l, P < 0.05) and decreased serum testosterone (19.4 to 11.5 nmol/l, P < 0.05) concentrations, yet there were no treatment effects on any of the other hormones. Glucose rates of appearance (R-a) and disappearance (R-d) were lower, and glycerol R-a-to-R-d ratio was not affected by E-2 administration. O-2 uptake, CO2 production, and respiratory exchange ratio were not affected by E-2; however, there was a decrease in heart rate (P < 0.05). Plasma lactate and glycerol were unaffected by E-2; however, glucose was significantly higher (P < 0.05) during exercise after E-2 administration. We concluded that short-term oral E-2 administration decreased glucose R-a and R-d, maintained plasma glucose homeostasis, but had no effect on substrate oxidation during exercise in men.