The isolation and characterization of human herpesvirus-6 (HHV-6) has been hindered by the lack of cell lines useful for its rapid propagation. Recently, we have reported that the MRC-5 cell line (human diploid lung fibroblasts) was susceptible for HHV-6 infection. In this study, we report on the isolation of HHV-6 from the peripheral blood or buffy coat of three chronic fatigue syndrome patients, one post-liver transplant patient, and one severe chronic active Epstein-Barr virus syndrome patient using the MRC-5 cell line. Additionally, it was observed by Southern blot hybridization studies that four of five isolates had different restriction enzyme fragment patterns than the isolate obtained from the National Institutes of Health with Eco RI. These data suggest the usefulness of the MRC-5 cell line in the isolation and characterization of HHV-6 from various patients.