In this paper, the characterization of four human malignant glioma cell lines is described. The four lines are positive for glial fibrillary acidic protein (GFAP) in variable amounts. One of them, LN 992, is positive for S-100 protein. Myelin basic protein could not be detected in any of the four lines. The four lines had high levels of CNPase activity. The karyotype shows polyploidy for all lines, with modal numbers ranging from 80 to 120 and various numbers of marker chromosomes. Particular attention has been paid to the surface phenotype and a panel of three antiglioma monoclonal antibodies (Mabs), five antimelanoma Mabs, one anti-CALLA Mab, and two anti-HLA-DR Mabs has been used in an antibody-binding radioimmunoassay for the four cell lines. Lines LN 215 and LN 235 are positive with two antiglioma Mabs, LN 992 is negative. The four lines are positive with all five antimelanoma Mabs, except for LN 992 which ist negative with Mab D5. LN 992 and LN 215 are positive with the anti-CALLA Mab N2A12. LN 308 and LN 992 are positive with anti-HLA-DR Mab D4-22. There was no correlation between the in vitro morphology of the lines and the expression of the various biochemical or surface markers. These results stress the heterogeneity of the phenotype of human malignant glioma lines. These lines will be useful tools for further immunologic studies.