To determine the relative roles of insulin intermediates in the glycolytic pathway in the control of lipogenesis, we have evaluated lipid synthesis in freshly isolated and primary cultures of hepatocytes from normal ad libitum fed and 72 hr fasted rats. In freshly isolated hepatocytes from fasted rats the rate of lipid synthesis is markedly depressed and resistant to the stimulatory effects fo insulin and spermine, a post insulin binding mimicker of insulin action. This is true whether lipid synthesis is evaluated from 3H2O or from (14C)-acetate. These characteristics are retained in primary cultures of these hepatocytes. This cell system, therefore, is suitable to evaluate the effects of insulin and substrates on the recovery of lipid synthesis in hepatocytes from fasted rats. Glucose, fructose, glycerol, pyruvate, and acetate in the incubation medium for 20 hr were without effect on the rate of lipid synthesis and insulin responsiveness in the primary cultures from fasted rats. However, when the cells were incubated for 20 hr with insulin, 10(-9) M, the rate of lipid synthesis and insulin responsiveness were comparable to that of primary cultures of hepatocytes from fed rats. The present studies demonstrate the unique role for insulin in restoring lipid synthesis and insulin responsiveness in mammalian liver from fasted animals.