This paper describes the development of monoclonal antibodies generated against hepatitis A virus (HAV). Monoclonal antibodies (MCABs) from two murine hybridoma cell lines were found to bind to an epitope recognized in the sera of patients recovering from infection with HAV. Ascites fluids containing MCABs from one hybridoma (H1 C19) inhibited a maximum of 70% of the 125I-labeled polyclonal human anti-HAV from binding to HAV-antigen in a competitive radioimmunoassay, indicating that the MCAB recognizes a major epitope of HAV. Monoclonal anti-HAV that was coated onto polystyrene beads was as effective as polyclonal antibodies in capturing HAV antigen from extracts of human feces, marmoset liver, and cultured cells. Radiolabeled MCAB was used to screen sera for anti-HAV. A collection of 117 sera was tested for total anti-HAV by competitive radioimmunoassay utilizing either 125I-labeled human polyclonal or mouse monoclonal antibody. Thirty-four specimens were similarly reactive by both systems, while the remainder were negative. Likewise, 28 specimens were similarly positive for IgM anti-HAV, and 12 specimens were negative using each of the two labeled antibodies. The data show that anti-HAV induced during disease is directed against a common major epitope of HAV and that MC anti-HAV can be used effectively as a reagent to detect these antibodies.