Ethanol has been shown to induce the activity of glucose-6-phosphate dehydrogenase (G6PDH). To clarify the mechanism behind this induction, we examined the role of acetaldehyde (AA), the first product of ethanol metabolism. In primary adult rat hepatocytes maintained in chemically defined medium, we examined the effect of AA on G6PDH activity, mRNA levels and lipid synthesis. We observe a 40% increase in G6PDH activity and a similar increase in mRNA levels, following exposure to 100 microM AA. The increase in activity was found to be maximal at 24 h while mRNA levels increased over controls as early as 3 h. The induction in G6PDH by AA was found to occur at lower concentrations and earlier time points than those reported using ethanol. The role of insulin, a known inducer of G6PDH activity was studied alone and in combination with AA on both G6PDH activity and mRNA levels as well as lipid biosynthesis. Insulin (300 ng/ml) was found to increase G6PDH activity, mRNA levels and [14C]-acetate incorporation into lipid. It was also shown to have an additive effect with AA on G6PDH activity, suggesting their actions are mediated via different mechanistic pathways. No change in [14C]-acetate incorporation into lipid, however, was observed with acetaldehyde alone.