The results obtained in a study of seroprevalence by means of ELISA and immunoblot with crude larval extracts of Anisakis simplex using 1008 human sera from Spanish people showing no clinical suspicion of anisakidosis are given. For the evaluation of the results obtained by ELISA the Diagnostic Index (DI) was used, as the ratio between the optical density resulting from the test serum and the optical density of the negative control. Forty-seven sera showed DIs between 1.5 and 2, and 14 sera were greater than 2. After comparison of the immunoblot analysis with the immunorecognition pattern of a human anisakidosis reference serum, a diagnostic criterion could be established for those sera that, at a 1/100 dilution, showed a DI by ELISA greater than 1.5. Seven of 14 selected sera with DIs in ELISA higher than 1.3 showed anti-Anisakis specific IgE antibodies by RAST fluoroimmunoassay.