Tumors of the nervous system were induced in Sprague-Dawley and Long-Evans rats by weekly administrations of 6 mg/kg N-methyl-N-nitrosourea in the drinking water. Three of these tumors, a grade 2 mixed glioma, a grade 2 to 3 astrocytoma and a grade 1 to 2 oligodendroglioma, were established in culture and propagated in vitro. The mixed glioma strain (75SD-G-376) and the astrocytoma line (75SD-G-420) were repeatedly subcultured, cloned at passage 90 and 120 and designated as 75SD-G-376C and 75SD-G-420C clone, respectively. The growth rate of the oligodendroglioma cell strain (77LE-G-180) was very low and the cells died off after the 5th in vitro passage. The glial nature of all lines was ascertained by demonstrating the presence of the S-100 protein in the culture cells. 2 1/2 years after the establishment in vitro of the 75SD-G-376 and 75SD-G-420 primary cultures, mass cultures as well as clones derived from them are still producing S-100 and thus are clearly comparable to the primary cultures, at least in this respect. From a morphological standpoint based on light microscopy, cells of clonal lines with relatively few and short processes differ, however, from cells of primary cultures and their uncloned lines. Therefore, the cell morphology of these clones can be viewed upon as a form of adaptation to the in vitro conditions. It can be concluded that permanent cell lines with well-defined properties can be grown from experimental brain gliomas successfully established in culture and maintained in vitro.