We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.