The oncofetal antigen (HGR-Ag) in brain tumors was newly defined using the monoclonal antibody derived from tissue extracts of an anaplastic astrocytoma. The HGR-Ag was purified through sequential chromatography using diethylaminoethyl-Sephadex A-50 and Con-A Sepharose affinity columns. The latter method indicated that HGR-Ag is not a glycoprotein, and Western blot analysis indicated a molecular weight of 80-90 kd. Purified antigen was used to produce antibody. This new antibody, designated H1H2, was shown to be an immunoglobulin G1 by immunodiffusion assay. Histological/immunohistochemical studies using the H1H2 antibody on paraffin and frozen tissue sections showed HGR-Ag to be intracellular rather than membrane-associated. The immunoreactivity of H1H2 was highest in malignant astrocytomas, glioblastomas, and normal fetal brain tissue. Neurinomas and certain carcinomas of other organs showed lesser, variable reactivity to H1H2. The strength of the antigen-antibody binding was correlated with the degree of malignancy in human gliomas. H1H2 bound to fetal brain tissue and undifferentiated neural tumors, but not to normal adult brain tissue, so HGR-Ag is probably a fetal oncoantigen.