Human leptomeningeal cells grown in culture were immortalized via transfection with an SV40 large T antigen gene construct under the control of the Rous sarcoma virus promoter. This cell line, designated LTAg2B, maintained the polygonal morphology characteristic of primary cultures, and stained positively in early passage for cytokeratin, a specific marker for arachnoid cells of the leptomeninges. Additional immunofluorescent staining revealed that these cells express vimentin and desmoplakin as well; these antigens have been found together only in normal arachnoid tissue and in meningiomas, which are the neoplastic derivatives of leptomeningeal cells. Significantly, LTAg2B cells demonstrate a greatly increased life span and growth rate relative to primary cultures. The establishment of this cell line should thus facilitate studies on the cellular and molecular biology of leptomeningeal cells, as well as elucidate their roles in certain pathological situations involving the leptomeninges, such as meningitis and meningioma tumor formation.