ABSTRACT. It is not known if milk antibody protects infants from herpes simplex virus (HSV) infection. As a first step to test this hypothesis, anti-HSV antibodies were studied in human milk. Paired serum and milk samples were analyzed for anti-HSV antibodies by ELISA, Western blot analysis (WBA), neutralization (NT) plaque assay, and antibody-dependent cellular cytotoxicity (ADCC) assay. Nineteen of the 20 serum samples showed anti-HSV activity by ELISA and ADCC, and 18 showed activity by WBA and NT. We found a significant association between the immunoassays for detection of anti-HSV antibodies in sera. Fewer of the human milk samples showed anti-HSV activity; only one milk sample was positive by ELISA and one by N T assay, four by ADCC and 12 by WBA. The milk sample from the seronegative donor was also negative. We found a poor association of antibody titers in human milk and serum antibody titers using ELISA, NT, and ADCC assays. There was a significant (p = 0.022) association between serum and milk results using WBA. Among the four assays, WBA was the most sensitive for antibody detection. It will be used in an on-going prospective study to determine the role of anti-HSV antibody in the protection against HSV infections in infants. (Pediatr Res 31: 591-595,1992)