A panel of 45 well-characterized monoclonal antibodies (MAbs) reactive to glycoprotein B (gB) of herpes simplex virus (HSV) type 1 was tested by ELISA and in antiviral functional assays (that included virus neutralization, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated lysis), using type 1 or 2 virus strains. All MAbs were ELISA-reactive. Eleven of the MAbs mediated neutralization and 9 mediated ADCC. All of the ADCC epitopes were contained within the amino-terminal half of the extracellular portion of gB. The ADCC reactions were strictly type 1-specific, whereas 9 of 11 neutralizing MAbs exhibited type-common activity. There was some association between the ADCC and neutralization activities, since of 12 MAbs with functional activity, 8 were positive in both assays. These results suggest that differences in the presentation of gB, and perhaps HSV-1 gB versus HSV-2 gB, on free virus and virus-infected cells determine epitope availability.