The in vitro secretion of cytomegalovirus (CMV)-specific antibodies by peripheral blood mononuclear cells (PBMC) was analyzed in patients with acute CMV infection and CMV-seropositive patients infected with human immunodeficiency virus. This active and spontaneous in vitro secretion was not affected by the depletion of T cells or adherent cells. The number of anti-CMV antibody-secreting cells was estimated to be 28-176/10(6) PBMC; 30%-65% of the total in vitro antibody production was CMV-specific. This secretion seemed to be independent of in vitro polyclonal B cell activation, in vitro antigen-specific lymphocyte stimulation, or in vivo Epstein-Barr virus-induced B cell transformation. In vitro anti-CMV antibody production may therefore result from an in vivo stimulation of the immune system by CMV antigens and might indicate CMV replication in the host.