A highly active fraction that was mitogenic for astroblasts but which contained no amino acids was identified during the purification of peptides from chick embryo brains. This material was purified by ultracentrifugation, ultrafiltration through Diaflo PM-30 and YM-2 membranes and retention on Diaflo YC-05, followed by ion exchange chromatography and reversed phase high performance liquid chromatography (HPLC) on a C18 Deltapak column. On thin layer chromatography and HPLC the material co-chromatographed with authentic commercially-obtained GMP. Its ultraviolet absorption spectrum was also identical with that of GMP. 1H and 31P nuclear magnetic resonance spectra of the isolated material were identical with those of GMP. The close match between the fast atom bombardment (FAB) mass spectra of the unknown material and authentic GMP indicated that the unknown material was GMP of molecular weight 363 Da. Authentic, commercial GMP stimulated the growth of cultured chick astroblasts in the same dose-dependent manner as the material from chick embryo brains; maximal stimulation was at 50 microM. Guanosine, GDP, and GTP also stimulated cell proliferation. The nucleotides were equally as effective as guanosine. 5'-Guanylyl imidodiphosphate, guanosine 5'-O-(2-thiodiphosphate), and guanosine 5'-N-(3-thiotriphosphate), guanine nucleotides which are relatively resistant to enzymatic hydrolysis, were also mitogenic, indicating that the nucleotides do not need to be degraded to nucleosides to be active and that they probably act extracellularly. Guanine nucleosides and nucleotides promoted astroblast growth when other growth factors were removed from the culture medium. The mitogenic effects of guanosine and its nucleotides were inhibited in a dose-dependent fashion by micromolar concentrations of theophylline, a characteristic of phenomena mediated by purinergic receptors. Guanosine and its nucleotides are released in micromolar concentrations by hypoxic or dying cells. Under these circumstances these compounds may stimulate division of adjacent cells in vivo.