A newly developed system (as described in the previous paper) employing separated herpes simplex virus (HSV) polypeptides electrophoretically transferred to nitrocellulose paper, Western Blot Analysis (WBA), was compared to microneutralization (MN) for the detection of type-specific antibody to HSV types 1 and 2 in 98 human sera. Sera containing HSV1-specific antibodies reacted at least twofold more densely (as quantitated by densitometric scanning) to HSV1 than HSV2 polypeptides, while sera with HSV2-specific antibodies reacted twofold more densely to HSV2 polypeptides. The reliability of this system was determined (1) by analysis of sera from patients with true primary herpes simplex infections, (2) by adsorption studies that removed cross-reacting antibodies, and (3) by comparison with neutralization. Of 36 sera found to have HSV1-type-specific antibody by WBA, 83% had MN ratios of less than 85 (indicating HSV1 antibody), while of 28 sera found to have HSV2-type-specific antibody by WBA, 89% had MN ratios of greater than 85 (indicating HSV2 antibodies). The MN ratio obtained on sera felt to have both HSV1-type-specific and HSV2-type-specific antibody by WBA ranged from 67 to 160. WBA agreed more closely with clinical isolates and adsorption studies than MN. This system not only accurately determines the HSV-type-specific antibody status of patients but provides important information on the specific immunogenic proteins.