The clinical features and the molecular epidemiology of primary herpes simplex virus type 1 (HSV-1) infection among children younger than 3 years of age were investigated in day-care nursery. Serial sera were assayed for anti-HSV-1 glycoprotein B antibody by enzyme-linked immunosorbent assay. Serologic examinations revealed 55 cases of primary HSV infection during the observation period. Fifty-one of them (93%) had typical herpetic gingivostomatitis, showing a high rate of clinically overt infection. Four outbreaks of herpetic gingivostomatitis were observed during the observation period. Forty-one children were infected with HSV-1 in the outbreaks. The rates of infection in the susceptible children were 81%, 73%, 78%, and 100%, respectively, in the four outbreaks. Restriction endonuclease analysis of DNA of isolated HSV revealed that only one strain of HSV-1 had been transmitted among children for a long period.