DNA sequences encoding glycoprotein B (gB) derived from herpes simplex virus type 1 (HSV-1) strain KOS321 were transferred to HSV-1 ANG. In cotransfection experiments the cloned HSV-1 KOS BamHI G fragment served as donor, and genomic DNA of two ANG variants as recipients. One of these variants, HSV-1 ANG path, expresses gC and the other, C18, was a spontaneous gC-negative mutant. Both ANG strains are of the syncytial (syn) phenotype whereas HSV-1 KOS321 is non-syncytial (syn+). Recombinants were identified by means of a monoclonal antibody which selectively recognizes gBKOS. Among the HSV-1 ANG path/gBKOS recombinants, the majority displayed an altered plaque morphology, i.e. they were of the syn+ phenotype. In contrast all of the C18/gBKOS recombinants were of the syn phenotype. The possibility that the mutant C18 carries a syn mutation not present in the parental strain could be excluded. Marker transfer experiments involving subfragments of the gB gene mapped the syn mutation of HSV-1 ANG path to a locus within the gene that has been previously termed syn 3. Subclones of HSV-1 ANG path were established either directly or after intermittent transfection or cotransfection with the KOS BamHI G fragment. The pathogenicity in mice of these clones was compared. The data obtained indicated that at high frequency, the BamHI G fragment confers apathogenicity.