Two isolates of human herpesvirus 7 (HHV-7) were recovered from phytohemagglutinin-activated peripheral blood mononuclear cells of a patient with chronic fatigue syndrome and of a healthy blood donor. A genetic polymorphism between the two isolates was detected by Southern blot analysis using a novel HHV-7 genomic clone (pVL8) as a probe. We developed optimized conditions for the in vitro propagation of HHV-7 by using enriched populations of activated CD4+ T lymphocytes derived from normal peripheral blood, resulting in the production of high-titered extracellular virus (> 10(6) cell culture infectious doses/ml). Bona fide syncytia formation was documented both in normal CD4+ T lymphocytes and in the Sup-T1 CD4+ T-cell line following infection with high-titered HHV-7. To identify neutralizing antibodies to HHV-7, a syncytia-inhibition test was developed. Variable titers of syncytia-neutralizing antibodies were detected in all the human sera tested, thus confirming the high prevalence of HHV-7 in the human population.