Female Sprague-Dawley rats were treated with a single ip dose of aflatoxin B1 (AFB1) (3 or 6 mg/kg). Twenty-four hours later and weekly until killed, some of the rats treated with AFB1 were given ethynylestradiol (EE) by gavage at the dose of 13 mg/kg. One, three, six, and nine months following the beginning of the experiment, animals were killed. Light microscopy of liver and histochemical determinations of gamma-glutamyltransferase (GGT) as well as the measurement of hepatic drug-metabolizing enzyme activities were investigated. The results show that AFB1 induced only very weak changes in the levels of different constituents studied. Thus, the mycotoxin did not affect GGT activity and increased epoxide hydrolase activity by a maximum of 42%. In contrast, EE significantly and progressively decreased (20 to 50%) the activity of UDP-glucuronosyltransferase (UDPGT) as well as the concentration of cytochrome P-450 and microsomal proteins. However, the estrogen increased the activity of epoxide hydrolase up to 150% as well as the activity of the hepatic (400%) and plasma (175%) GGT. The results also indicate that AFB1 amplified the EE-induced increase in liver weight and enhanced the depressive effects of the estrogen on microsomal proteins, cytochrome P-450, and UDPGT. Foci of cellular alteration which consisted of clear, acidophilic and basophilic cell lesions were seen in the livers of treated rats examined by light microscopy. These lesions were more prominent in the livers of animals given combinations of AFB1 and EE; they were accompanied by a strong intensity of GGT staining in the periportal area and a marked increase of the enzyme activity in the plasma (324%). From the sixth month, the livers of some animals treated with the combinations of AFB1 and EE showed hyperplastic nodules. This study indicates that the interaction between chronic administration of EE and a single ip injection of AFB1 induces hepatic lesions considered as possible forerunners of liver cell carcinomas. It also shows that GGT is a potential marker of preneoplastic lesions and may be used, therefore, in epidemiologic surveys in humans exposed to liver carcinogens such as the aflatoxins.