Herpes simplex virus type 1 (HSV-1) used for vaccine production was isolated from a nasal recurrent infection and propagated over a limited number of passages in human diploid cells only. It was designated as HSV-1 BW3 and has been characterized by neutralizing antibodies as a typical HSV type 1 strain. In vitro transformation studies performed with this isolate in mouse or hamster cells revealed only very low, if any, transforming capacity. A preparation of HSV-1 BW3 which can be used as seed lot for vaccine production has been proven to be free of any adventitious agents such as bacteria, fungi mycoplasma or viruses other than HSV-1 BW3. An envelope antigen (EAG) preparation was obtained from purified HSV-1 particles. It was free of detectable viral DNA and could be proven to be efficacious in mouse models against challenge infections with either HSV-1 or HSV-2. The potency of the vaccine was greatly enhanced by the addition of poly-inosinic-poly-cytidylic acid complexed with poly L-lysine and carboxymethylcellulose (PICLC) as adjuvant. A single vaccine dose was sufficient to protect mice from morbidity and the fatal outcome of HSV infection, but not from the establishment of latency. Persistent ganglionic infections could be, however, significantly reduced by repeated administration of the vaccine before primary infection.