A critical determinant for intraperitoneal virulence of Herpes Simplex Virus Type 1 (HSV-1) strain F has been ascribed to the BamHI-B fragment of the genome. This region contains two nonessential genes, UL55 and UL56, of unknown function. To investigate the contribution of these genes to viral pathogenesis, we have generated two recombinant HSV-1 viruses, inUL55 and inUL56, in which the corresponding gene was inactivated in strain F by insertion of an ICP6::lacZ cassette. Strains inUL55 and inUL56, which did not express the UL55 and UL56 RNA, respectively, were virulent in mice inoculated intraperitoneally. Strains inUL55 and inUL56 also established latent infections in the trigeminal ganglia of mice inoculated via the cornea. These results demonstrate that the HSV-1 UL55 and UL56 genes are not critical for intraperitoneal virulence or establishment of latent infection.