Protein induces an increase in insulin concentrations when ingested in combination with carbohydrate. Increases in plasma insulin concentrations have been observed after the infusion of free amino acids. However, the insulinotropic properties of different amino acids or protein (hydrolysates) when co-ingested with carbohydrate have not been investigated.The aim of this study was to define an amino acid and protein (hydrolysate) mixture with a maximal insulinotropic effect when co-ingested with carbohydrate.Eight healthy, nonobese male subjects visited our laboratory, after an overnight fast, on 10 occasions on which different beverage compositions were tested for 2 h. During those trials the subjects ingested 0.8 g*kg(-)(1)*h(-)(1) carbohydrate and 0.4 g*kg(-)(1)*h(-)(1) of an amino acid and protein (hydrolysate) mixture.A strong initial increase in plasma glucose and insulin concentrations was observed in all trials, after which large differences in insulin response between drinks became apparent. After we expressed the insulin response as area under the curve during the second hour, ingestion of the drinks containing free leucine, phenylalanine, and arginine and the drinks with free leucine, phenylalanine, and wheat protein hydrolysate were followed by the largest insulin response (101% and 103% greater, respectively, than with the carbohydrate-only drink; P <0.05).Insulin responses are positively correlated with plasma leucine, phenylalanine, and tyrosine concentrations. A mixture of wheat protein hydrolysate, free leucine, phenylalanine, and carbohydrate can be applied as a nutritional supplement to strongly elevate insulin concentrations.