In order to examine the pathways of hepatic glycogen repletion in the periportal and perivenous zones of the liver, [1-13C]glucose (99% enriched) was infused intraduodenally into conscious, 24-h fasted rats for 3 h. The liver was then quickly perfused in situ, and the cytoplasmic contents of the periportal and perivenous hepatocytes were selectively sampled by modification of the dual-digitonin-pulse technique (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). The 13C isotopic enrichment at each carbon position of the glucosyl units of hepatic glycogen was determined by 13C NMR and that of the C-1 position by gas chromatography-mass spectroscopy. From comparison of hepatic glycogen repleted by direct incorporation of plasma glucose (glucose----glucose-6-P----glucose-1-P----UDP-glucose----glycogen) was calculated to be 29% in the periportal zone and 35% in the perivenous zone, assuming equal glycogen synthetic rates within the two zones. Thus, the majority of glycogen is derived by an indirect route (glucose--------3-carbon unit--------glucose --------UDP-glucose--------glycogen) in both the periportal zone and in the perivenous zone. In conclusion, in a 24-h fasted rat there does not appear to be a major difference between the periportal and perivenous hepatocytes in the percent of glycogen synthesized by the direct pathway following a glucose load.