An optimized assay for herpes simplex virus type 1- and 2-induced deoxythymidine kinase (dTk) is described which used [125I]iododeoxyuridine (IUdr) as a substrate. Values for Km and Vsat were determined for both viral and cellular dTK, using either deoxythymidine or IUdR as a substrate. A comparison between the two substrates revealed that higher reaction velocities and lower Km values were obtained with IUdR. A standard assay was designed which uses 10(-7) M IUdR as a substrate. This assay can detect herpes simplex-induced dTK from as few as two infected cells and is several orders of magnitude more sensitive than conventional dTK assays which use 10(-5) M dT as a substrate. An easily detectable blocking activity, which was shown to be mainly confined to the immunoglobulin G antibody class, was found in most human sera which were positive for complement-fixing antibody against herpes simplex virus.