The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro, but a limited range of mitogens have been defined. We investigated whether three identified neonatal SC mitogens [glial growth factor (GGF), platelet-derived growth factor BB (PDGF-BB), and basic fibroblast growth factor (bFGF)] are required to stimulate mitosis of adult SCs. Adult SCs were isolated from mouse sciatic nerves by mechanical and chemical dissociation, following three experimental steps: 1) culturing the dissociated cells for 24 hr in 10% FCS-F12 medium, 2) culturing these cells in serum-free medium for the next 48 hr, and 3) purifying adult SCs by differential adhesion. We describe a new method for preparation of SCs from peripheral nerves of adult mouse that provides 99.5% pure SCs populations at cell yields of greater than 3 X lo3 cells/mg of starting nerve wet weight within 5 culture days. Although mitosis of SCs in culture in response to mitogens requires the presence of serum, the complex nature of serum renders difficult a complete analysis of mitogens required for SCs DNA synthesis, so we examined the proliferating response of adult SCs to GGF, PDGF-BB, and bFGF in serum-free medium. GGF alone had mitogenicity for adult SCs in a dose-dependent manner, and synergistic activation coupling with forskolin was not observed. Neither PDGF-BB nor bFGF was mitogenic for adult SCs when used alone or with forskolin. Measurement of intracellular cyclic AMP levels in SCs cultured with these growth factors showed that cAMP levels were not changed by GGF and bFGF, and PDGF-BB reduced the cAMP level to half of basal one. This study demonstrated that adult SCs could proliferate without serum factors and forskolin in response to GGF, and adult SCs were activated by GGF through a signal transduction pathway separate from the cyclic AMP-dependent system. In addition, PDGF-BB or bFGF has no mitogenic effect on adult SCs under serum-free conditions. 0 1995 WiIey-Liss, Inc.