The etiology of multiple sclerosis (MS) remains unknown, but there are indications of a role of human herpesvirus 6 (HHV-6), especially variant A, in the pathogenesis. Higher serum antibody reactivity against an HHV-6 early protein, p41, has been found in MS cases than in controls. The antigen, however, was purified from infected cells with a monoclonal antibody also reactive with a protein (p38) likely to be of cellular origin. To avoid serological crossreactivity with the cellular protein, recombinant p41 proteins from HHV-6A strain GS and HHV-6B strain Z29 were expressed as glutathione-S-transferase fusion proteins (p41-GST), and used as antigens in an enzyme-linked immunosorbent assay (ELISA). p41 variant specific monoclonal antibodies reacted strongly with the respective recombinant proteins. Serum IgM and IgG reactivities with the recombinant p41 antigens were analysed in patients with manifest MS, patients with optic neuritis, patients with other neurological diseases, and in one group of healthy controls. All sera were HHV-6 IgG seropositive by immunofluorescence. The serum IgM or IgG reactivities against the recombinant p41 antigens did not differ significantly between the groups, and the reactivities against the variant A and B antigens were identical. In many samples, the reactivity was very low. The results indicate that p41 is not an optimal target for HHV-6 serology studies, and that the data obtained with the p41 antigen prepared from infected cells (possibly including also p38) should be interpreted with caution. J. Med. Virol. 66:394-399, 2002. © 2002 Wiley-Liss, Inc.