The presence or absence of latent multiple infections with herpes simplex virus (HSV)-1 was investigated. DNA cleavage patterns resulting from the digestion of viral DNA by restriction enzymes were studied. The DNA cleavage patterns of asymptomatically shed viruses isolated from the saliva of subjects at different time were compared to HSV-1 isolated from patients with HSV-1 infections of the oral cavity. If a patient had HSV-1 isolated from their saliva more than two times, the asymptomatically shed viruses were used in these experiments. This criterion was met by 13 patients. All patients underwent the viral isolation procedure on separate occasions. In addition, 55 viral strains from 10 patients with HSV infections were included in the experiment. The HSV-1 lesions were caused by a reactivation of the same strain of virus. The asymptomatically shed HSV-1 viruses had identical DNA cleavage patterns in only 4 of 13 patients (31%). The viruses isolated on separate occasions from the remaining 9 patients (69%) had different DNA cleavage patterns. Several investigators have reported the existence of latent HSV multiple infections.