A lethal graft-versus-host disease (GVHD) was induced in cyclophosphamide (Cy)-treated C3H mice by i.v. injections of C57 bone marrow (20, 30, or 50 X 106) or spleen cells (5 X 106) which had been pretreated in vitro withVibrio choleraeneuraminidase (VCN) or heat-inactivated VCN. The surviving C3H recipients were grafted with C57 skin to evaluate the presence of tolerance or immunity to the donor cells. The acute lethality of high doses (400 mg/kg) of cyclophosphamide was prevented equally well by VCN-treated or unmodified allogeneic bone marrow cells. The acute lethal GVHD, attributable to high doeses (400 mg/kg) of cyclophosphamide and allogeneic spleen cells, could be induced equally well by VCN-treated and unmodified spleen cells. The results suggest that VCN-treated cells maintain their viability, can reconstitute cyclophos-phamide-treated mice, and can induce GVHD in severely immunosuppressed mice. At more moderate doses of cyclophosphamide (300–350 mg/kg), VCN treatment of donor bone marrow and spleen cells delayed the onset and reduced the lethality of the GVHD when compared with cells treated with heat-inactivated VCN. Surviving animals who had received donor cells treated with heat-inactivated VCN were frequently tolerant of donor (C57) skin grafts. Surviving mice who had received VCN-treated cells were immune to the donor skin grafts. VCN treatment appears to render cells less tolerogenic than normal so that mice do not become tolerant and the GVHD cannot develop. The results are compatible with our previous findings that VCN increases the immunogenicity of normal lymphoid cells, both in unmodified hosts and in vitro.