Liver sections from aging ad libitum-fed and diet-restricted B6C3F1 male mice were evaluated immunohistochemically for pi-class glutathione S-transferase (GST-II). GST-II immunostaining of hepatocytes was diffuse and occurred in periportal regions of hepatic acinus, whereas perivenous areas were weakly stained or were stain-free. Expression of GST-II was significantly diminished in diet-restricted mice in all age groups and was associated with a marked decrease in liver tumor development. As most spontaneous liver tumors were GST-II positive, it can be speculated that they developed from GST-II positive initiated hepatocytes. To determine whether dietary restriction induces apoptosis in GST-II-positive hepatocytes, 24-month-old ad libitum-fed mice were introduced to 40% diet restriction. After 1 week of diet restriction, a decrease in GST-II expression was associated with a threefold increase in the frequency of apoptotic bodies as detected by terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling of DNA fragments. A two-step immunohistochemical procedure revealed that approximately 70% of apoptotic bodies were GST-II positive. These results suggest that spontaneous, potentially preneoplastic hepatocytes in tumor-prone B6C3F1 mice are eliminated by apoptosis with dietary restriction.