Infant formulas are often heat sterilized in hospitals where water contamination or nosocomial infection is a concern, but there are few studies of the effect of high heat on the nutritional value of infant formula. In particular, the effect of heat sterilization on free amino acid (FAA) concentrations is seldom discussed. In view of the importance of these nutrients for infant growth, we investigated the FAA concentrations of infant formula after heat sterilization. Powdered infant formulas were reconstituted with hot water (801C) in glass bottles and placed in an autoclave for 5 min at 1051C and 5600 kg/m 2 of pressure. Additional samples of formula were prepared by conventional methods to serve as controls. After autoclaving, we measured the FAA concentrations with ion exchange chromatography. The results were compared with those obtained after conventional preparation. We found a 19.5% lower amount of total protein after autoclaving compared with conventional preparation. Concentrations of total FAA were significantly lower after autoclaving (696.57101.4 vs 899.47152.2 mmol/l, P ¼ 0.01). The concentrations of individual amino acids were also lower in autoclaved infant formulas, with differences ranging from À4.1 to 71.5% (mean 22.6%). Concentrations of certain amino acids were more than 30% lower, such as valine (71.5%), citrulline (61.1%), glutamine (60.6%), ethanolamine (54%), and lysine (39.2%). Both essential and nonessential amino acids were similarly affected by autoclaving, 28.17 and 27.13%, respectively, lower than in controls (P ¼ 0.37). The concentration of ammonia was significantly higher after autoclaving (645.2776.2 vs 393.27140.7 mmol/l, P ¼ 0.0003). However, the urea level was significantly lower after autoclaving than after conventional preparation (1110.87162.7 vs 1426.57209.5 mmol/l, P ¼ 0.0004). The accumulation of ammonia may reflect degradation of protein and amino acids. Autoclaving clearly results in decreased concentrations of FAA in infant formula. The increased concentration of ammonia after autoclaving is of concern if it leads to deleterious effects.