The acute effect of selective hyperglycemia or hyperinsulinemia on late gestation fetal ovine glucose transporter protein (GLUT-1, GLUT-3, and GLUT-4) concentrations was examined in insulin-insensitive (brain and liver) and insulin-sensitive (myocardium and fat) tissues at 1, 2.5, and 24 h. Hyperglycemia with euinsulinemia caused a two- to threefold increase in brain GLUT-3, liver GLUT-1, and myocardial GLUT-1 concentrations only at 1 h. There was no change in GLUT-4 protein amounts at any time during the selective hyperglycemia. In contrast, selective hyperinsulinemia with euglycemia led to an immediate and persistent twofold increase in liver GLUT-1, which lasted from 1 until 24 h with a concomitant decline in myocardial tissue GLUT-4 amounts, reaching statistical significance at 24 h. No other significant change in response to hyperinsulinemia was noted in any of the other isoforms in any of the other tissues. Simultaneous assessment of total fetal glucose utilization rate (GUR_f) during selective hyperglycemia demonstrated a transient 40% increase at 1 and 2.5 h, corresponding temporally with a transient increase in brain GLUT-3 and liver and myocardial GLUT-1 protein amounts. In contrast, selective hyperinsulinemia led to a sustained increase in GUR_f, corresponding temporally with the persistent increase in hepatic GLUT-1 concentrations. We conclude that excess substrate acutely increases GUR_f associated with an increase in various tissues of the transporter isoforms GLUT-1 and GLUT-3 that mediate fetal basal glucose transport without an effect on the GLUT-4 isoform that mediates insulin action. This contrasts with the tissue-specific effects of selective hyperinsulinemia with a sustained increase in GUR_f associated with a sustained increase in hepatic basal glucose transporter (GLUT-1) amounts and a myocardial-specific emergence of mild insulin resistance associated with a downregulation of GLUT-4.