To determine alpha-fetoprotein (AFP) immunogenicity in vivo, the presence of antibodies in sera of 60 hepatocellular carcinoma, 15 liver cirrhosis, and 15 chronic hepatitis patients was evaluated by Western blotting and immunoprecipitation analyses using purified human AFP. High titers of anti-AFP immunoglobulins were detected in 14 hepatocellular carcinomas (P = 0.0006), 3 liver cirrhosis (P = 0.0173), and 1 chronic hepatitis patient, but they were not detected in 40 healthy individuals. Therefore, spontaneous immune responses to AFP are significantly associated to liver diseases (P = 0.0015). Patient immunoglobulins recognized proteic linear epitopes that were cryptic in the native protein, as demonstrated by their restricted reactivity with denatured deglycosylated AFP. Thus, in pathological liver conditions, tolerance to this self-molecule is circumvented. The identification of AFP immunogenic epitopes may contribute to defining novel immunotherapeutic strategies targeting this antigen.