The responses of serum immunoreactive insulin (IRI), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and total liver lipid to starvation and refeeding were studied in male Wistar rats. All parameters studied were reduced by starvation. In rats refed a high-carbohydrate diet for 2 days, G6PD, ME, and total liver lipid rose to over twice the ad-libitum-fed values; IRI was approximately at or below ad-libitum-fed values. Treatment of rats with 8-azaguanine prevented the overshoot of G6PD, ME, and total liver fat, but did not prevent return to ad-libitum-fed values. A second cycle of starvation refeeding induced an even greater overshoot of G6PD and ME, while the overshoot of total liver lipid was not different from that observed after the first starve–refeed cycle, and serum insulin levels were again normal. Treatment with 8-azaguanine during the second refeeding only allowed G6PD and ME to reach approximately twice ad-libitum-fed values. Two basic conclusions were drawn from the data: (1) that hyperlipogenesis in starved–refed rats is not due to continuous hyperinsulism, and (2) that the newly formed RNA responsible in part for the enzyme overshoot is relatively stable.