The pentose phosphate pathway (PPP) is widely assumed to play a key role in both reductive biosynthesis and protection from oxidative stress because it is the major source of NADPH. However, little is known about the activity of the PPP in fatty liver which is characterized by both oxidative stress and lipogenesis. This study was designed to test whether the PPP is active in parallel with lipogenesis and antioxidant processes in the fatty liver of whole animals. Eight- and 16-week old, obese Zucker diabetic fatty rats and their lean littermates received [U-13C3]glycerol, and 13C labeling patterns of glucose and triglycerides were analyzed for the assessment of hepatic PPP activity and the potentially-related processes simultaneously. Oxidative stress, antioxidant activity, and NADPH-producing enzymes in the liver were further examined. Both PPP activity and lipogenesis increased in the fatty liver of young obese Zucker rats, but decreased together in older obese Zucker rats. As expected, lipid peroxidation measured by malondialdehyde increased in the fatty liver of obese Zucker rats at both ages. However, evidence for antioxidant processes such as [glutathione] or activities of glutathione reductase, glutathione peroxidase and catalase was not altered. Hepatic PPP activity paralleled lipogenesis, but was dissociated from biomarkers of oxidative stress or antioxidant processes. In summary, NADPH from the PPP was presumably consumed for reductive biosynthesis rather than antioxidant defense in the fatty liver.