Fatty acid synthase (FAS) expression is low in liver and adipose tissue of suckling rats and increases markedly after weaning on to a high-carbohydrate low-fat diet. It has been shown previously that glucose alone, via an increase in intracellular glucose-6-phosphate level, stimulated the accumulation of FAS mRNA in cultured white adipose tissue of suckling rats. The regulation of FAS expression by glucose and hormones (insulin, dexamethasone and triiodothyronine) was studied in cultured hepatocytes from suckling rats. In hepatocytes cultured for 48 h in the absence of hormones and glucose, FAS mRNA, as well as glucokinase mRNA, levels remained undetectable. Glucose alone was unable to stimulate FAS expression. The combination of hormones, in the absence of glucose, has a marginal effect on FAS mRNA levels. However, FAS mRNA levels were increased in the presence of both glucose and the combination of hormones. This demonstrated that the hormonal induction of FAS mRNA was dependent on the presence of glucose in the culture medium. We have then investigated if glucokinase expression could be a prerequisite for the stimulation of FAS expression in response to glucose. Hepatocytes were cultured for 48 h in the absence of glucose but in the presence of insulin, dexamethasone and triiodothyronine. In these conditions, glucokinase mRNA and activity were markedly increased but there was no accumulation of FAS mRNA. When these hepatocytes were then exposed to various levels of glucose, FAS mRNA rapidly accumulated. Glucose stimulation of FAS expression was observed only in hepatocytes which expressed glucokinase activity. The importance of glucokinase expression for the induction of FAS mRNA by glucose is supported by the striking correlation between glucose-6-phosphate concentrations and the levels of FAS mRNA. This study clearly demonstrates that: (a) glucose metabolism is directly involved in the regulation of FAS gene expression; (b) the effect of hormones is partly due to their capacity to induce in the hepatocytes the capacity for glucose phosphorylation.