Impaired cardiac glucose metabolism and glucose transport have been shown in the insulin resistant fatty Zucker rat. The aim of the present study was to examine the translocation of the insulin-sensitive glucose transporter (Glut4) in the heart of the fatty Zucker rat under in vivo conditions. Insulin was injected into both lean (FA/?) and fatty (fa/fa) Zucker rats via the tail vein. The time course of cardiac Glut4 translocation was studied by determining the subcellular distribution of Glut4 using a newly developed ELISA quantitation method. Insulin (10 U/kg) caused a 30% and 37% increase in plasma membrane Glut4 content at 20 min after injection in lean and fatty rats respectively. The plasma membrane Glut4 contents in the basal and insulin-stimulated states were significantly lower in the fatty rat when compared to the lean control. The dose effect of insulin (2.5-10 U/kg) on Glut4 mobilization to the plasma membrane was similar in both phenotypes. The time course of Glut4 mobilization to the plasma membrane (5-30 min), which was similar in both lean and fatty Zucker rats, showed that maximal translocation was reached at 5 min post insulin injection and persisted throughout the remaining 25 min. However, in fatty Zucker rats, Glut4 content in the intracellular membrane remained unchanged at all insulin doses and all time points studied. Collectively, these results show that Glut4 recruitment to the plasma membrane is responsive to insulin in the fatty Zucker rat heart and that the maximal response was similar to that in lean Zucker rats. However, the recruitment of Glut4 to the plasma membrane was not associated with changes in the intracellular membrane Glut4 content.