Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.