The effect of different hormones and growth factors was assayed on the in vitro growth and enzymatic activities of 2',3'-cyclic nucleotide 3'phosphohydrolase (CNP) and glutamine synthetase (GS) of rat glioma C6 cells at two different passages in culture. Young cultures (passage 26), mainly oligodendrocytic, and older cultures (passage 134), predominantly astrocytic, were treated with 10 microM dexamethasone, 20 ng/ml transforming growth factor alpha (TGF alpha), 10 ng/ml insulin, 20 ng/ml platelet-derived growth factor (PDGF), and 20 ng/ml, epidermal growth factor (EGF) in serum-free chemically defined media. In vitro growth rate was measured in terms of DNA content, by a fluorometric method of diaminobenzoic acid, and rate of DNA synthesis by 3H-thymidine incorporation. CNP activity (marker for in vitro oligodendrocytes) and GS activity (marker for astrocytes) were determined spectrophotometrically. Dexamethasone reversibly and significantly inhibited growth of C6 glioma in early and late passages. PDGF and insulin promoted in vitro growth only in late passage but not in early passage cells, whereas EGF and TGF alpha did not significantly affect growth. An increase in CNP activity was observed in early passage cells under the effect of PDGF and insulin. The increase in GS activity induced by insulin and dexamethasone suggests a differentiating role for these factors in C6 glioma cells. These results further present the C6 glioma cell line as a useful model for studies on glial cell properties and responsiveness in culture and support its use in experimental aging in vitro.