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Polymerase chain reaction methods are becoming the standard for the diagnosis of herpes simplex virus (HSV) encephalitis. Little is known, however, about the stability of HSV DNA in cerebrospinal fluid (CSF) specimens. Our results demonstrate that HSV DNA is extremely stable in CSF specimens containing lymphocytes and monocytes. We observed little DNA degradation in specimens stored for 30 days at room temperature (20–23°C), refrigerator temperature (2–8°C), or freezer temperatures (- 18 to −22°C and −69 to −72°C). Specimen storage conditions are therefore not so critical for HSV encephalitis detection as for other viral illnesses. It is therefore not necessary to perform a second lumbar puncture to obtain a fresh specimen for the detection of HSV DNA.