In a blind study, 14 isolates of herpes simplex virus type 1 (HSV-1) that included nine isolates from a temporal cluster of HSV infections in a hospital Pediatric Intensive Care Unit and five unrelated isolates were analyzed by digestion of their DNA with four restriction endonucleases. These enzymes (Hsu1, Bgl11, EcoRl, and Hpal) cleave the DNA in about 52 sites. To date, at least 16 sites have been found to be variable in the sense that they may be present or absent independently of any other cleavage site. This characteristic is stable, and no change was observed on serial propagation of the strains in culture or following repeated isolation, as long as 12 years apart, from humans. Analyses of the isolates readily discriminated between those belonging to the temporal cluster of hospital infections and the unrelated strains. They also showed that there were two independent introductions of HSV-1 into the Pediatric Intensive Care Unit resulting in two clusters of epidemiologically related infections. This type of analysis has the potential of becoming a powerful tool for tracing the spread of HSV-1 and very likely of other herpesviruses in the human population.