A model was developed for in vivo study of the human glioma-derived D-54 MG cell line in the brains of immunosuppressed Fischer 344 rats. The rats were injected with horse anti-rat thymocyte serum before and after intracerebral inoculation with 5 or 10 microliters of a D-54 MG tumor cell suspension. Reproducible mortality distributions were obtained, with deaths occurring 18 to 34 days after intracerebral inoculation. Tumors grew as well circumscribed intracerebral masses with sheets of anaplastic cells, areas of necrosis bordered by concentrated nuclei, and minimal lymphocytic infiltration. Cytogenetic analysis revealed the same general chromosome distribution and markers in the heterotransplanted glioma cells as in the cultured line. Blood-brain barrier disruption was demonstrated by intracerebral tumor staining after intravenous injection of Evans blue dye. The in vivo growth of D-54 MG in immunosuppressed rats provides a reliable experimental model for the study of chemotherapy, immunodiagnosis, and immunotherapy of a human glioma-derived tumor in an animal sufficiently large to evaluate intracarotid or intratumoral injection of therapeutic agents.