Glucose may be incorporated into glycogen both by an indirect pathway that involves the metabolism of glucose to C₃ intermediates prior to incorporation into glycogen and by a direct mechanism that utilizes the sequence glucose → glucose-6-P → glucose-1-P → UDP-glucose → glycogen. Studies were carried out to determine the major pathway in primary cultures of rat hepatocytes. When cells were incubated with medium containing [3-³H]- and [¹⁴C(U)]-glucose the ratio of ³H/¹⁴C in glycogen was 70–80% of that of the glucose in the medium. This ratio was unaffected by increases in glucose concentration or insulin, both of which promoted large increases in the incorporation of glucose into glycogen. Relative ³H/¹⁴C ratios in glycogen of 25–30% were observed when [2-³H]- and [¹⁴C(U)]-glucose was employed; this ratio was doubled in the presence of 2-deoxyglucose or sorbitol, each of which inhibits phosphohexose isomerase. It is concluded that about 75% of the glucose undergoes isomerization between glucose-6-P and fructose-6-P, while about 25% is further glycolysed to C₃ intermediates, prior to incorporation. Lactate added to the medium was incorporated into glycogen to an extent of only 20% of that of glucose. However, the presence of lactate resulted in a large increase in the incorporation of glucose into glycogen. Little net deposition of glycogen was observed in these studies. It is concluded that cultured hepatocytes may be a model for the fed organism, and in this condition the direct pathway for the incorporation of glucose into glycogen was predominant.