Antigen- or lectin-stimulated T lymphocytes and human T-cell leukemia virus (HTLV)-infected cell lines secrete lymphokines that can influence the growth and function of a variety of cell types. We recently demonstrated that supernatants from the HTLV-II-infected Mo-T-cell line stimulate the proliferation of rat brain oligodendrocytes and astrocytes. We have now purified a glial growth-promoting factor (GGPF) from these supernatants. Purification from serum-free conditioned medium was accomplished by sequential concentration, ammonium sulfate precipitation, lentil-lectin affinity chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. GGPF is assayed by its ability to stimulate DNA synthesis in oligodendrocytes, as measured by [3H]thymidine uptake. The purified GGPF has an apparent M(r) of 30,000 when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, however, GGPF appears as a single band of M(r) 18,000. Both reduced and unreduced forms have biological activity, suggesting that GGPF exists in both a functional monomeric and dimeric form. Purified GGPF appears to be a biochemically and functionally distinct lymphokine.