The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs. These results suggest that the mechanisms of the effects of insulin on the glucokinase and L-type pyruvate kinase mRNAs are partially the same, although they are largely different. Dexamethasone alone tended to decrease the levels of the two mRNAs, but enhanced their induction by insulin. Thyroid hormone had a permissive effect on the induction of glucokinase mRNA, but not on that of L-type pyruvate kinase mRNA, by insulin. 8-(4-Chlorophenylthio)-cAMP inhibited the induction of the two mRNAs by insulin.