The most direct approach to elucidating the roles of herpes simplex virus (HSV) proteins in the viral replicative cycle has been to isolate temperature-sensitive, cytolysis-resistant, and drug-resistant mutants that exhibit alterations in the synthesis or activity of these proteins. The development of procedures for the introduction of temperature-sensitive mutations into physically defined regions of the viral genome and for fine mapping of these mutations has proven especially valuable. Thus, (1) hydroxylamine mutagenesis of the HSV-1 BglII I fragment (coordinates 0.312-0.415) has facilitated the genetic and functional characterization of the gene for the major viral DNA-binding protein of 130 K molecular weight; (2) the selection of a mutant conditionally able to render infected cells resistant to immune cytolysis has led to identification of an HSV gene involved in the processing of viral glycoproteins; and (3) the combined use of temperature-sensitive and drug-resistant mutants has led to a better definition of the physical limits and functional domains of the gene for HSV DNA polymerase.