In Italy epizootics of equine influenza often occur, but no virus isolation has been reported since 1971. This paper describes the antigenic and biochemical characterization of two equine influenza viruses isolated in Italy from 1985 to 1989. The virus isolates were shown to differ antigenically from earlier strains of the same subtype, A/equine/Miami/1/63 (H3N8). Monoclonal antibody analysis showed that the haemagglutinins of these strains were serologically indistinguishable from A/equine/Fontainebleau/1/79, a variant of A/equine/Miami, never isolated in Italy before. One of the two virus isolates was obtained from a horse immunized with a bivalent inactivated influenza vaccine, not containing A/equine/Fontainebleau/79 antigens. The vaccine failure underlines the importance of antigenic relatedness between currently circulating viruses and vaccine strains. Therefore, to improve the protection afforded by equine immunization, the vaccine composition should be decided according to the results of a virological surveillance activity, systematically conducted among horses.