We have shown previously that prolonged exposure to insulin and glucose impairs the insulin-responsive glucose transport system in primary cultured adipocytes. To assess the ability of insulin and glucose to regulate other cellular insulin actions, epididymal rat adipocytes were cultured in media containing 0-15 mM D-glucose and with or without insulin (50 ng/ml). After 24 h, cells were washed and basal and maximally insulin-stimulated rates of 2- deoxy-D-glucose uptake, L-leucine incorporation into protein, glucose oxidation to CO2, glucose incorporation into lipids, and glycogen synthase activity were measured. The results confirmed that glucose potentiates insulin's chronic ability to decrease basal and maximal glucose transport rates by ~50% at 5 mM glucose and by ~70% at 15 mM glucose compared with control cells. However, neither glucose nor insulin, alone or in combination, affected rates of leucine incorporation into protein. In addition, basal and maximal rates of glucose oxidation and of glucose incorporation into lipids were not regulated by glucose, and maximal responses declined ~50% over 24 h only when insulin was not present during preincubation (i.e., chronic insulin exposure was necessary to maintain full maximal responses). Glycogen synthase activity was measured in a cell-free system (0.5 mM UDP-glucose, with 10 or 0.01 mM glucose-6-phosphate) after exposing intact cells to glucose and insulin. Both short-term (1 h) and long-term (24 h) exposure to glucose alone led a dose-dependent increase in I-form and D-form glycogen synthase activity. Chronic exposure to insulin also increased total glycogen synthase activity (I-plus D-form) but did not affect absolute rates of maximally stimulated I-form activity. Glucose (but not insulin) increased the cellular content of immunoreactive glycogen synthase by 70% after 1 h. These results show that 1) chronic exposure to glucose and insulin impairs insulin responsiveness of the glucose transport system but does not affect rates of amino acid incorporation into protein; 2) the chronic presence of insulin is necessary for the maintenance of normal maximally stimulated rates of glucose oxidation and of glucose incorporation into lipids in cultured cells; and 3) glucose increases both D-form and I-form glycogen synthase activity, in part by increasing the amount of synthase protein, whereas chronic insulin exposure increases total glycogen synthase activity without altering maximal absolute rates of I-form activity. In conclusion, glucose and insulin differentially regulate the multiple biological actions of insulin in cultured adipocytes, indicating that underlying mechanisms involve the hormone effector systems or components of signal transduction unique to individual action pathways. In NIDDM, defects in glucose transport but not other insulin actions may be acquired as a result of high circulating blood glucose and insulin concentrations.