The beta-adrenergic system is involved in the control of energy metabolism and expenditure. The beta2-adrenergic receptor (beta2-AR) gene shows polymorphisms that have been associated with obesity in several studies. In vitro and in vivo studies suggest differences in beta2-AR-mediated function between these polymorphisms. The aim of this study was to investigate the influence of genetic variation in codon 16 of the beta2-AR gene on energy metabolism in humans. Thirty-four subjects were recruited [Gly16Gly (n = 13), Gly16Arg (n = 16), or Arg16Arg (n = 5)]. The beta2-AR was stimulated with two doses of salbutamol (50 and 100 ng/kg fat-free mass per minute) after blockade of the beta1-adrenergic receptors with atenolol. Energy expenditure and plasma substrate and hormone concentrations were measured. The increase in energy expenditure (DeltaEE) was significantly different among groups in which the Arg16Arg group showed the lowest increase (P < 0.05 vs. Gly carriers). In a multiple regression model, variations in the increase in nonesterified fatty acid concentration during salbutamol infusion (partial r = 0.51) and the polymorphism contributed significantly to the variation in DeltaEE. Thirty-five percent of the variation in DeltaEE was explained by these two factors. We conclude that subjects with the Arg16Arg polymorphism of the beta2-AR gene have a reduced thermogenic response to beta2-adrenergic stimulation. Although this relatively small study needs confirmation, the findings support a role for this polymorphism in the development and maintenance of overweight and obesity.