A primed constant infusion of [6-3H]glucose was used to determine glucose turnover in rats fasted for 48 h that had previously been pair-fed isoenergetic 4% and 14.4% protein diets for 6 wk. The protein-depleted rats had a low body weight and lower plasma glucose and plasma insulin concentrations compared with the 14.4% protein pair-fed control animals. The rate of appearance of glucose in plasma in the protein-depleted rats was significantly less than that in the pair-fed controls. During an intravenous infusion of unlabeled glucose (38.8 mumol.kg-1.min-1) plasma glucose concentrations and glucose appearance increased in both groups. Plasma insulin concentration remained lower in the 4% protein-fed group though the percentage of increase above basal was similar in both groups during glucose infusion. The clearance of glucose from the blood and suppression of endogenous glucose production during the exogenous glucose infusion were similar in both control and protein-depleted rats. Rats fed a 14.4% protein diet ad libitum consumed more diet than the pair-fed groups and their body weights were greater. However, fasting plasma glucose and insulin, concentrations were similar to the 14.4% protein pair-fed animals. This indicates that the observed changes in glucose metabolism in the 4% protein-fed animals were due to a reduced dietary ratio of protein to carbohydrate rather than reduced energy intake.