Many individuals who are seronegative for one member of the human Herpesviridae family are strongly seropositive for other members. Using sera from such individuals, the radioimmunoassay technique demonstrated absence of antigen-antibody cross-reactions between varicella-zoster virus (VZV) and herpes simplex virus (HSV) at levels of less than one part in 1,000. Sera containing antibody to both HSV and VZV were absorbed with antigens of one agent without significantly altering the amount of remaining antibody to the other antigen. This further suggests that HSV and VZV do not share a common antigen. The same radioimmunoassay technique and serum absorption method that revealed no serological cross-reactions between HSV and VZV revealed the expected cross-reaction between HSV type 1 (HSV-1) and HSV-2. Reciprocally absorbed anti-HSV-1 and anti-HSV 2 sera were able to differentiate the two HSV types reliably. No cross-reactions were seen between cytomegalovirus, VZV, and HSV or between Epstein-Barr virus, VZV, and HSV. We postulate that heterotypic antibody responses sometimes observed for VZV after primary infections by HSV may not be due to shared antigens, but to activation of latent VZV infections, release of new VZV antigens, and consequent stimulation of new antibody production to VZV.