Monoclonal antibodies (mAbs) specific for human herpesvirus-6 (HHV6) proteins were derived from the splenocytes of mice immunized with HHV6 TAN isolate-infected peripheral blood mononuclear cells. The two mAbs 8C8 and 7C7 reacted by means of immunofluorescence and immunoperoxidase assays with both variant A and variant B isolates giving two different staining patterns. In infected cells, cytoplasmic diffuse staining was observed with mAb 8C8, whereas intense nuclear staining was obtained with mAb 7C7. These different locations of viral target proteins were confirmed by confocal microscopy. The mAb 8C8 reacted with a family of six glycoproteins designated as the gp72 complex in the case of variant A strains and gp63 complex in the case of variant B strains. The endoglycosidases H and F reduced those glycoproteins to a putative precursor molecule of 58 kDa. The mAb 7C7 reacted with 116 and 109 kDa proteins with the two HHV6 variants. These two mAbs did not neutralize virion infectivity in the absence of complement. No cross-reactivity was observed when these mAbs were used in immunoperoxidase assay and immunoblotting against the proteins of human cytomegalovirus or other human herpesviruses. Thus, the two mAbs 8C8 and 7C7 may be valuable tools for the diagnosis and biological investigation of HHV6 infections.