We report that recombinant glia maturation factor (GMF), a 17-kD brain protein, can be phosphorylated in vitro at the serine residue by protein kinase C (PKC), protein kinase A (PKA), and casein kinase II (CKII), and at the threonine residue by p90 ribosomal S6 kinase (RSK). Endogenous GMF in astrocytes is phosphorylated at the serine (major) and threonine (minor) residues within 15 min after stimulation by phorbol 12-myristate 13-acetate (PMA). Phosphorylation gradually subsides over the next 24 h. The increased phosphorylation is not blocked by the protein synthesis inhibitor cycloheximide and is not accompanied by a rise in the mRNA for GMF and is therefore strictly a posttranslational regulatory phenomenon. The rapid and transient phosphorylation of GMF upon cellular activation suggests an intracellular role, possibly with involvement in signal transduction.