Woodchuck hepatitis virus infection of the eastern woodchuck represents a useful model for the study of hepatitis B virus infection and disease in humans, including hepatocellular carcinoma. In man, hepatocellular carcinoma is frequently detected and monitored using assays for serum alpha-fetoprotein. To study the relationship between α-fetoprotein and woodchuck hepatitis virus-induced hepatocellular carcinoma in the woodchuck model, we produced a monoclonal antibody to woodchuck α-fetoprotein and used biophysical and immunochemical methods to demonstrate its specificity and affinity (7 × 108 L/mol) for woodchuck α-fetoprotein. A competition radioimmunoassay was then developed and standardized for measuring serum α-fetoprotein concentrations. In the radioimmunoassay system, woodchuck α-fetoprotein was detected between 20 ng/ml (20% to 25% inhibition) and 8,500 ng/ml (97% to 98% inhibition). Elevated serum α-fetoprotein concentrations (450 to 452,000 ng/ml) were measured in 21 of 23 woodchucks in the advanced stages of woodchuck hepatitis virus-induced hepatocellular carcinoma. Serum α-fetoprotein was elevated above normal ( ≥ 450 ng/ml) as early as 3 to 11 mo before terminal hepatocellular carcinoma in 11 of 16 of the woodchuck hepatitis virus-carrier woodchucks. In a pilot study, serum α-fetoprotein became markedly elevated above normal in woodchuck hepatitis virus-carrier woodchucks that developed hepatocellular carcinoma but not in serologically recovered or uninifected woodchucks (i. e., without hepatocellular carcinoma). Thus, α-fetoprotein may provide a useful nonivasive marker in the woodchuck model for detecting and monitoring woodchuck hepatitis virus-induced hepatocellular carcinoma from earlier stages.(HEPATOLOGY 1990; 11:824-833.)