In the absence of a truly representative animal model, the question of whether EBV-related diseases can be prevented by a vaccine has been studied for the first time in humans. A live recombinant virus based on the licensed vaccinia strain Tien Tan, expressing under the 11K vaccinia promoter the major EBV membrane antigen BNLF-1 MA (gp 220-340), was constructed and tested in three different human populations: EBV-positive and vaccinia-virus-exposed adults; EBV-positive, non-vaccinia-virus-exposed juveniles; and EBV and vaccinia virus-naive infants. No significant titre variations for EBV were observed in the adults, but EBV-neutralising titres increased in the vaccinated juveniles, while antibodies to VCA of EBV remained unchanged. All nine vaccinated infants developed antibodies to MA (membrane antigen) with neutralising properties in vitro; three of these infants were infected by EBV via natural routes over a period of 16 months after vaccination and all ten unvaccinated control infants became infected. It has been shown for the first time that protection against and/or delay of EBV infection by the natural route is possible in humans and that live vaccinia vectors can be used and are efficacious.