Three monoclonal antibodies (MAbs 1051a, 1051b and 1051c) were raised against a surface region (residues 597-686) of the herpes simplex virus type 1 (HSV-1) DNA polymerase (HSV pol), and their epitopes were mapped. The MAbs reacted serotype specifically with the native and denatured HSV pol, as shown by Western blot analysis, immunoprecipitation and immunofluorescence microscopy, indicating their usefulness for biochemical studies and clinical diagnosis of HSV-1 infections. MAb 1051c, displaying the least cross-reactivity with cellular proteins in the Western blot analysis, was successfully utilized not only for coimmunoprecipitation, but also for the analysis and three-dimensional modeling of the cellular sites of HSV pol interaction by confocal laser immunofluorescence microscopy.