The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 micrograms mouse-1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3-9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76-118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.