A reliable and reproducible method for determining specific reactivity to herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in human sera has been developed. Human sera were used to immunoprecipitate HSV-specific glycoprotein antigens from both HSV-1- and HSV-2-infected cell extracts. The viral glycoproteins precipitated from these extracts were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect specific reactivity of the sera with distinct type-specific antigens of HSV-1, HSV-2, or both as well as with type-common glycoprotein antigens. By examining a large number of human sera, this method was found to be more reliable than the standard microneutralization test in discriminating between single-positive (positive for HSV-1 or HSV-2) and double-positive (positive for HSV-1 and HSV-2) sera.