Hyperinsulinemia was required to preserve normal intravenous glucose tolerance in late pregnancy in the rat (18 to 21 days' gestation). To evaluate the site and mechanism of this insulin resistance, insulin binding and action were measured in hepatocytes freshly isolated from control and gravid animals. As expected, glucose concentrations were lower, insulin levels were higher, and hepatocyte size increased in the pregnant animals. Insulin binding was similar in liver cells from the two groups. No difference was found in sensitivity or responsiveness of insulin-stimulated net 14C-glucose incorporation into glycogen in the hepatocytes from the control and gravid rats. These results suggest that the well-documented, but ill-defined, insulin antagonism of late pregnancy does not include the liver and is a postreceptor defect, most likely residing in muscle tissue.