A rapid multisite radioimmunoassay for measurement of human alpha-fetoprotein (AFP) that uses two high-affinity monoclonal antibodies directed against distinct and separate determinants on the protein was developed and designated M-RIA. The sensitivity of the "simultaneous-sandwich" M-RIA is approximately equal to 0.5 ng/ml of serum after a 1-hr incubation period. Serum AFP levels have been measured in 1747 individuals with hepatocellular carcinoma (HCC), acute and chronic hepatitis B virus infection, chronic hepatitis B surface antigen (HBsAg)-carrier states, cirrhosis, other malignant tumors, and normal and disease controls to determine the specificity of the assay. Eighty percent (68/85) of patients with HBsAg-positive HCC had AFP levels of greater than 200 ng/ml (range, 260 to greater than 200,000 ng/ml). In contrast, all 450 normal subjects and 477 chronic HBsAg-positive carriers had levels of less than 20 ng/ml. More importantly, in acute and chronic hepatitis B, cirrhosis, and other malignant tumors and in the remaining disease controls, AFP levels were less than 20 ng/ml in 99.3% of the subjects, the great majority (greater than 96%) being less than 5 ng/ml. Indeed only two of 1635 individuals, one with acute hepatitis and the other with carcinoma of the esophagus had AFP levels of greater than 100 ng/ml. These observations are at variance with previous studies with conventional polyvalent RIAs of AFP levels of greater than 20 ng/ml in approximately equal to 40% of acute and chronic hepatitis and in 30% of cirrhosis. This striking specificity of the M-RIA is probably due in part to recognition of epitopes unique to AFP and suggest that such an assay may be used in the detection, early identification, and monitoring of AFP-producing tumors in high-risk populations.