Herpes simplex virus (HSV) is the prototype of a family of large, enveloped, double-stranded DNA viruses, the Herpesviridae, which cause significant morbidity and mortality in humans. Productive replication of HSV in cells in culture results in definitive changes in cellular physiology and metabolism, ultimately leading to lysis. These definitive aspects of viral-host interactions enable diagnosis of HSV infections. In this unit, a series of methods are described for the propagation, quantification, and storage of HSV. Infectious center assays are used to measure the titers of HSV stocks. In addition, immunological methods are described for documenting the accumulation of viral polypeptides in infected whole cell extracts, as well as in situ using indirect immunofluorescence. These techniques should be beneficial to basic research virologists utilizing standard laboratory HSV strains, as well as clinical microbiologists interested in characterizing HSV isolated from patients.