The prognosis of herpes simplex encephalitis (HSE) depends on the early and appropriate administration of specific antiviral therapy. We retrospectively reviewed 38 cases of children with proven HSE, to evaluate the reliability of polymerase chain reaction results, according to the time of cerebrospinal fluid (CSF) sampling. Initial negative results were observed in 8 of 33 CSF samples drawn before day 3 of the disease and were significantly associated with a low level of protein and <10 leukocytes/mm3 in the CSF.