One of the few spontaneous gliomas in inbred animals, the VM/Dk spontaneous murine astrocytoma (SMA), has seen limited use. Previously restricted to an in vivo system, the SMA was only transplantable intracerebrally (IC) using nonquantifiable suspensions of normal brain and tumor tissue. Prior atempts at establishing permanent tumorigenic SMA cell lines have not succeeded; tumorigenicity was lost during serial in vitro passage. We have established three different cell culture lines from a serially IC-transplanted SMA and two from tumors that arose from intraperitoneal (IP) injection of the IC-transplanted SMA. In contrast to previous cell cultures and transplantable lines of SMA, all five cell lines are not only tumorigenic IC but subcutaneously (SC) as well. Astrocytic features are present in three of five lines to varying degrees, evidenced by in vitro and in vivo morphology, response to dibutyryl cyclic AMP (db-cAMP), and the presence of neuroglial fibers: None of the lines express CNPase, S-100, or GFA proteins in significant amounts. P560, highly tumorigenic and possessing the most astrocytic features of the five lines, extends the use of the spontaneous astrocytoma system of the inbred VM/Dk mouse strain by allowing quantitative in vivo and in vitro experiments.