This study compared the rate of isolation of herpes simplex virus (HSV) from >36000 samples of mucosal secretions obtained from 296 HSV-infected persons versus the rate of detection of HSV DNA, by means of a real-time quantitative polymerase chain reaction (PCR) assay. Overall, HSV was isolated in 3.0% of samples, and HSV DNA was detected in 12.1% of samples. The mean number of HSV DNA copies was 10(4.9) in samples obtained on days when HSV lesions were present and 10(4.4) in samples from days when HSV lesions were absent. There was a linear relationship between the ability to isolate virus in culture and the log number of copies of HSV DNA in the sample; this relationship persisted in samples from men or women, in samples from human immunodeficiency virus-negative or -positive participants, and in samples obtained on days when lesions were present or absent. In home-collected specimens, the ratio of PCR positivity to viral-culture positivity rose from 3.8:1 in the winter to 8.8:1 in the summer months, reflecting the lability of viral-culture specimens transported during warm weather.