Methods of labeling surface proteins on herpes simplex virus (HSV) which have minimal effect on the biological activity of the virus are useful for the study of both the localization and function(s) of surface proteins. The present work describes a procedure using a water-soluble biotin compound, sulfo-NHS-biotin, which is unable to penetrate biological membranes and reacts with primary amines in proteins. Labeled proteins were detected by binding of [125I]streptavidin. Specific reaction with surface proteins was shown in Western blots using antibodies against selected proteins in the envelope or in the tegument. Proteins susceptible to iodination were also biotinylated, but the efficiency of labeling varied from one protein to another. As a result of freezing and thawing of the virus, as well as the manipulations involved in Ficoll gradient purification, internal proteins were labeled. The infectivity of the virus was reduced by approximately 40% after biotinylation. Labeled viruses were visualized by fluorescein isothiocyanate-conjugated streptavidin, and seen as distinct spots on the surface of the cells.