Xenogeneic transplantation has recently become a subject of interest for the transplantation community due to the current organ shortage, which could be partially or even totally solved by the development of this strategy. However, xenogenetic rejection remains a formidable barrier preventing such use in a clinical setting. The spheroidal aggregate-cultured hepatocytes of WKA rats were injected into the spleen of C3H mice, and quantitative assessment of transplanted xenogeneic hepatocytes using 99mTc-GSA demonstrated that hepatocytes decreased dramatically 2 days after transplantation (day 2) and few viable hepatocytes in spheroids were detected on day 3. The NK activity significantly increased on day 1, and gammadelta receptor/FasL-expressing T cells appeared on day 2. These results suggested that xenogeneic cytotoxicity consisted of gammadelta T cells through the Fas/Fas ligand system, as well as non-T-cell-mediated cellular response, in the MHC-unrestricted pathway in this intrasplenically transplanted xenogeneic hepatocyte model.