The effect of ethanol, alone and in combination with glucocorticoid and insulin, on glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Maintenance of hepatocytes from fasted animals in a culture medium devoid of hormones and ethanol resulted in a 2.5-fold increase in G6PDH activity in 48 hr. Parallel cultures treated with glucocorticoid and insulin or glucocorticoid, insulin and ethanol stimulated enzyme activity 6- and 9-fold, respectively in 48 hr. Treatment with ethanol for 48 hr potentiated basal and glucocorticoid plus insulin-induced enzyme activity 1.4-fold. The activity of G6PDH mRNA, estimated by cell-free translation of hepatic mRNA in a mRNA-dependent reticulocyte lysate and by RNA dot-blot hybridization, was compared with enzyme activity and relative rate of G6PDH synthesis. The increases in enzyme activity observed in response to glucocorticoid and insulin or ethanol, alone or in combination with glucocorticoid and insulin, were paralleled by comparable increases in the rate of synthesis and mRNA levels of G6PDH. The results of this study show that the glucocorticoids, insulin and ethanol interact to stimulate the synthesis of G6PDH primarily by increasing the concentration of G6PDH mRNA.