First-generation rats received either 20% (standard) or 8% (suboptimal) protein nutrition during pregnancy and lactation. Suboptimal protein nutrition led to reduced body weights of the second-generation progeny at day 19 of gestation (10%, p < 0.001) and at weaning (33% reduction, p < 0.001). Control (born of 20% protein-fed dams) and experimental (born of 8% protein-fed dams) offspring received 20% protein diet after weaning and were studied on day 19 of gestation at 9 to 12 weeks after weaning. Basal glucose turnover was lower (29%, p < 0.05) and glucose utilization by fast-twitch muscle, adipose tissue and diaphragm significantly reduced in experimental offspring. Hyperinsulinaemia increased whole-body glucose disposal rate in both control (2.3-fold, p < 0.001) and experimental (3.2-fold, p < 0.001) offspring. Hyperinsulinaemia normalised the suppression of glucose utilization observed in diaphragm, heart and adipose tissue, but not in fast-twitch muscle, where rates remained 30-40% lower in the experimental offspring. Glucose tolerance and insulin secretion after i.v. glucose were unimpaired in the pregnant experimental offspring. A 27% reduction in basal glucose utilization, without impaired growth, was observed for the third-generation fetuses of the experimental offspring. The results demonstrate that growth retardation evoked by suboptimal protein nutrition during early life leads to decreased basal glucose turnover and glucose utilization by a range of maternal tissues and the fetus during a subsequent pregnancy. It is not, however, associated with any major permanent impairment of glucose-stimulated insulin secretion or insulin action during pregnancy.