The activity of glucose-6-phosphate dehydrogenase (G6PD) is inhibited by the addition of polyunsaturated fat (PUFA) to a high carbohydrate diet. To define the regulated step, we measured enzyme activity, accumulation of G6PD mRNA, and transcriptional activity of the gene. At steady-state, G6PD activity and mRNA abundance were inhibited by 80% in the livers of mice fed a high-fat diet (6% safflower oil) compared to mice fed a low-fat diet (1% safflower oil). Inhibition of mRNA accumulation was 20% by 4 h and was maximal by 9 h after beginning the high-fat diet. Changes in mRNA accumulation preceded changes in enzyme activity, indicating pretranslational regulation. The rapid kinetics of G6PD mRNA accumulation depended on prior dietary history of the mice. In meal-trained mice, abundance of G6PD mRNA increased by twofold within 4 h of the onset of a low-fat meal and was maximal by 12 h. In contrast, an increase in G6PD mRNA was not observed until 12 h after refeeding starved mice and the increase was maximal (12-fold) by 27 h. Transcriptional activity of the gene was measured using the nuclear run-on assay. The G6PD probes were rigorously screened for repetitive elements and for transcription of the noncoding strand of the gene. Throughout the time course of changes in G6PD mRNA accumulation due to PUFA or refeeding, transcriptional activity of the gene did not change. Therefore, regulation of G6PD expression by nutritional status occurs at a posttranscriptional step.