After a primary human cytomegalovirus (HCMV) infection, antibody titer to human herpesvirus 6 (HHV-6) rises. To determine if this occurs because of simultaneous infection with both viruses, serologic responses to these viruses were investigated among healthy women who received a live HCMV vaccine or acquired HCMV from an infected child. Both vaccination and natural infection caused four-fold or greater titer rises to HHV-6. Analysis of sera from 5 children with primary HHV-6 showed no serologic response to HCMV. Absorption of 7 sera with HCMV antigens reduced titers to HHV-6 by two- to fourfold. Immunoblot analysis of sera obtained from 10 persons before and after a primary HCMV infection revealed that after HCMV infection, 2 persons developed IgG reactivity to the 116-kDa protein (gp116) of HHV-6 and to two cleavage products of this protein. Reactivity to HHV-6 gp116 appeared after HCMV seroconversion and was removed by absorption of sera with HCMV glycoprotein gB (gB), indicating that antibodies to HCMV gB cross-react with HHV-6 gp116, the likely gB homologue of HHV6. The serologic diagnosis of HHV-6 infection requires excluding a primary HCMV infection. © 1993 by The University of Chicago.