The metabolic state occurring with refeeding after fasting is characterized by the rapid restoration of hepatic glycogen. Recent evidence suggests that a main substrate for glycogenesis is lactate. Because adipose tissue is an active site of lactate production that increases with fasting, we examined the magnitude and duration of lactate production by isolated adipocytes from three adipose depots of rats fasted for 48 h and then refed for up to 96 h. The data show that 48 h of fasting results in a markedly elevated rate of adipocyte lactate production, which increased from 3-9% of total glucose metabolized in the fed state to 49-60% in the fasted state. During the refeeding period, lactate production remained elevated for 12-24 h and then declined. Mesenteric adipocytes had a higher rate and more prolonged elevation in lactate production than cells from the other two depots. We conclude that, with refeeding after a fast, adipocyte glucose conversion to lactate remains elevated during the time of hepatic glycogen restoration. This suggests that adipose tissue may actively produce lactate for glycogenesis during refeeding.