Experiments were designed to estimate the effect of in vivo hormonal treatment of rats on serine metabolism in isolated hepatocytes by incubating hepatocytes in the presence or absence of 3-mercaptopicolinic acid (a potent inhibitor of phosphoenolpyruvate carboxykinase), the relative flow of [14C]serine carbon to [14C]glucose via the serine dehydratase (SDH)-initiated vs. serine amino-transferase (SAT-initiated pathways could be estimated. Streptozotocin-induced diabetes caused a tripling of the absolute rate of [14C]serine conversion to [14C]glucose, along with a shift in the relative importance of the SAT-mediated pathway. Hydrocortisone treatment had no significant effect on either the rate or route of serine metabolism. The SAT-mediated pathway was the major route of serine conversion to glucose after 4 days of chronic glucagon injections, although the absolute rate of conversion was enhanced by only 50%. This was the only treatment examined in which SDH was not the major route for serine gluconeogenesis. The enzyme activity responses of SDH and SAT to hormonal manipulation previously reported do not necessarily reflect the observed changes in pathway flux reported in the present study.