Hormonal and substrate regulation of hepatic glycogen accumulation was evaluated in primary cultures of hepatocytes prepared from 1-day-fasted rats. Hepatocytes were cultured in media containing 5 mM-glucose and 10 mM-lactate and then exposed to 100 nM-dexamethasone for 4 h before an increase in glucose concentration and the addition of insulin. When this protocol was used to mimic the post-prandial state in vivo, net glycogen accumulation (over 2 h) and insulin (10 nM) effects were linear at physiological (5-10 mM) and supraphysiological (20-30 mM) glucose concentrations. To define the role of substrates in glycogen accumulation, hepatocytes were incubated in a buffered salt solution containing 10 mM-glucose and either 10 mM-lactate or 5 mM-glutamine, or both. In the absence of hormones, net glycogen accumulation was increased by 59%, 83%, and 127% by the addition of lactate, glutamine, and lactate plus glutamine respectively, compared with incubations with glucose alone, and 6-fold in the presence of substrates, insulin and dexamethasone. Labelling with [3-3H]glucose and [U-14C]glucose showed that in the absence of hormones approx. 50% of glycogen formation came from glucose via the direct pathway and the remainder from glucose via the indirect pathway or from non-glucose precursors, or both. Insulin-dependent enhancement of glycogen formation is through stimulation of both the direct and indirect pathways, and dexamethasone-dependent stimulation occurs through stimulation of both these pathways of glycogen formation from glucose as well as from non-glucose precursors. Lactate serves as a gluconeogenic C3 precursor for the observed enhanced glycogen formation, whereas glutamine-dependent enhancement of glycogen accumulation occurs primarily through a stimulation of the direct and indirect pathways of glycogen formation from glucose.