Rat liver glucose-6-phosphate dehydrogenase (G6PD) activity was studied in starved-refed rats given diets containing three levels of fat (35%, high-fat; 5%, low-fat; 0%, fat-free). Elution characteristics from DEAE-cellulose, Km for glucose-6-phosphate, pH optimum, and molecular weight appeared to be similar. During storage or heat denaturation, stability apparently was lowest of G6PD of livers from rats refed the high-fat diet. Heat stability was enhanced by the addition of NADP, but some differences due to diet persisted. Titration of a constant amount of enzyme with heating gave inconsistent results: in two of four experiments rats refed the high-fat diet had an equivalence point twice that of rats refed the fat-free diet. This difference disappeared if the antibody titration was carried out in the cold. The diet-induced instability of the G6PD, as measured in vitro, was reversible by changing the diet of the rats.