Isotopic measurements of protein kinetics are useful for the investigation of metabolic protein disorders during surgical illness. The effects of rate and route (oral vs parenteral) of nutritional substrate intake have not been well defined. Fischer 344 rats were infused with a total parenteral nutrition (TPN) solution at either 25, 100, or 175% of their normal substrate intake or were fed an oral diet ad libitum. After 4 days, [15N]glycine was infused at 0.138 mg 15N/hr for 24 hr. Whole-body protein turnover (WPT), synthesis, and catabolism were determined by 15N urea enrichment. Fractional synthesis rates (FSR) of liver and muscle protein were calculated by analyzing 15N tissue enrichment. WPT (r = 0.93, P less than 0.001) and liver FSR (r = 0.57, P less than 0.01) increased linearly with TPN infusion rates. All rats had protein synthesis rates greater than catabolism rates except for the rats infused with 25% TPN. Although caloric intake was the same in rats fed orally and those infused with 100% TPN, the orally fed rats had faster WPT (P less than 0.001), synthesis (P less than 0.05), catabolism (P less than 0.001), and liver FSR (P less than 0.05) than the TPN rats. Muscle FSR was not significantly affected by either the route of feeding or the TPN infusion rate. In this study, rate and route of substrate intake affected protein kinetics in the whole animal and liver, but not in muscle. Rate and route of nutrient intake need to be carefully specified and controlled during isotopic studies of protein kinetics.