Background. The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts.Methods. Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1(+) and CD4(+)/CD8(+) cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx: host mice.Results. A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection. with the m144 molecule or following antibody depletion of host NK1.1(+) cells and in beige host mice. Other studies support some role for CD8(+) but not CD4(+) cells.Conclusions. These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.