Ephedrine is used to help achieve weight control. Data on its true efficacy and mechanisms in altering energy balance in human subjects are limited. We aimed to determine the acute effect of ephedrine on 24-h energy expenditure, mechanical work and urinary catecholamines in a double-blind, randomized, placebo-controlled, two-period crossover study. Ten healthy volunteers were given ephedrine (50 mg) or placebo thrice daily during each of two 24-h periods (ephedrine and placebo) in a whole-room indirect calorimeter, which accurately measures minute-by-minute energy expenditure and mechanical work. Measurements were taken of 24-h energy expenditure, mechanical work, urinary catecholamines and binding of (±)ephedrine in vitro to human β₁-, β₂- and β₃-adrenoreceptors. Twenty-four-hour energy expenditure was 3.6% greater (8965±1301 versus 8648±1347 kJ, P< 0.05) with ephedrine than with placebo, but mechanical work was not different between the ephedrine and placebo periods. Noradrenaline excretion was lower with ephedrine (0.032±0.011 μg/mg creatinine) compared with placebo (0.044±0.012 μg/mg creatinine) (P< 0.05). (±)Ephedrine is a relatively weak partial agonist of human β₁- and β₂-adrenoreceptors, and had no detectable activity at human β₃-adrenoreceptors. Ephedrine (50 mg thrice daily) modestly increases energy expenditure in normal human subjects. A lack of binding of ephedrine to β₃-adrenoreceptors and the observed decrease in urinary noradrenaline during ephedrine treatment suggest that the thermogenic effect of ephedrine results from direct β₁-/β₂-adrenoreceptor agonism. An indirect β₃-adrenergic effect through the release of noradrenaline seems unlikely as urinary noradrenaline decreased significantly with ephedrine.