Background: Wound healing is a complex process, dependent on available nutrition substrates. When used together with -hydroxy -methylbutyrate, arginine and glutamine have been shown to increase collagen deposition in human subjects. However, there are no experimental investigations on the influence of this amino acid mixture with regard to secondary wound healing. The aim of this study is to investigate the effects of the supplementation of these 3 amino acids on the healing of open wounds in otherwise healthy animals. Materials and Methods: Twelve rats were divided into control and treatment groups. Two 2-cm x 1-cm full-thickness skin defects were prepared on each subject. The rats in both groups received a diet containing 1.2 g of protein per 100 g of body weight per day. The treatment group, in addition, received 200 mg/kg L-arginine, 200 mg/kg L-glutamine, and 40 mg/kg -hydroxy -methylbutyrate every day. Wound sizes were measured every 2 days. On the 10th day, tissue samples were taken for histopathologic evaluation and also for the measurement of hydroxyproline concentrations. Results: There was no statistically significant difference between mean wound sizes for the 2 groups (P > .05). There was also no statistically significant difference between the groups with regard to histological healing parameters (reepithelialization [P = 1.00], granulation tissue [P = 1.00], collagen accumulation [P = .455], inflammatory cell accumulation [P = .455], angiogenesis [P = .242]) or tissue hydroxyproline concentrations (P = .240). Conclusion: Diet supplemented with arginine, glutamine, and -hydroxy -methylbutyrate is not beneficial in enhancing secondary healing of open wounds in rats. Further research regarding this topic is warranted.