We have shown that mice infected with mouse hepatitis virus A59 (MHV-A59) develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). The autoAb recognized conformational as well as linear antigenic determinants in the enzyme, and the autoimmune response was not entirely restricted to molecular mimicry and/or epitope spreading. Since the N- and C-terminal portions of the enzyme were the most reactive with autoAb, the fine specificity of these Ab was investigated. Immobilized 15-mer linear peptides (overlapping by 14 amino acids) spanning the N-terminal FAH sequence 1-49 were recognized by Ab from MHV-infected mice. The pattern of reactivity indicated the existence of two major epitope cores (i.e., sequences 9-23 and 30-44), and sequence comparison permitted the identification of two minimal epitopes, DSDFPIQ (amino acids 9-15) and IGDQILD (amino acids 36-42). Mutational analysis of sequences 9-23 and 30-44 indicated that residues 9-12 (DSDF) from the first major N-terminal epitope, and residue 36 (I) from the second, were the key amino acids energetically important for Ab contact. Interestingly, those residues were inside the two minimal epitopes previously predicted. The C-terminal portion of the enzyme (sequence 390-419) presented only one major epitope, located between residues 390 and 409. In this case, the minimal epitope had nine amino acids, CQGDGYRVG, corresponding to the FAH sequence 396-404 that outlines a loop specific for the enzyme. Data indicated that neither the FAH minimal epitopes nor the key residues important for binding to Ab from MHV-infected mice have their counterparts in the viral proteins. However, location of the energetically important residues in the tertiary structure of the enzyme originates a virtual conformational epitope. Such hypothetical B-cell epitopes could be present in any viral protein, originating a cross-reaction leading to the autoimmune response induced by MHV.