Sera of 200 non-transfused healthy male blood donors were tested for antibody reactivity to cell-surface antigens of cultured astrocytoma cells. Positive reactions were observed only rarely by protein-A assay (PA), in about half the cases by immune adherence assay (IA) and in nearly all cases by anti-C3 mixed hemadsorption assay (C3-MHA). In general, titers were low and only seven sera showed reactivity at 1:1,000. Serum 537 showed the strongest reaction. The anti-astrocytoma reactivity in this serum was due to an IgG antibody. Extensive absorption analysis with a panel of cell lines and fresh cells of both benign and malignant origin, as well al fetal cells, revealed that this serum detected an antigen that was present on most neural-crest-derived tumors (astrocytomas, melanomas and neuroblastomas), on very few other malignant tumors and on fetal brain. The antigen detected by serum 537 shows close relationship to the astrocytoma antigen AJ which had been defined by the serum of a patient with astrocytoma. Both antigens appear to be differentiation antigens present predominantly on non-epithelial neoplasms. The antigen detected by serum 537 is heat-stable and pronase-resistant. The sera of two other healthy donors apparently had a similar specificity, whereas the four other high-titered sera and all other sera detected class-III antigens which were non-specific and not tumor-restricted.