Apotransferrin (aTf), intracranially administered into newborn rats, produces increased myelination with marked increases in the levels of myelin basic protein (MBP), phospholipids and galactolipids, and mRNAs of MBP and 2', 3' cyclic nucleotide 3'-phosphohydrolase (CNPase). Cytoskeletal proteins such as tubulin, actin, and microtubule-associated proteins are also increased after aTf injection. In contrast, almost no changes are observed in myelin proteolipid protein (PLP) or in its mRNA or cholesterol. In the present study, we used brain-tissue slices and cell cultures highly enriched for oligodendroglia to investigate signaling pathways involved in the action of aTf, and to find out whether cytoskeletal integrity and dynamics were essential for its action upon the neural expression of certain genes. Treatment of brain-tissue slices with aTf produced a marked increase in the expression of MBP, CNPase, and tubulin mRNAs. Colchicine, cytochalasin, and taxol severely reduced the effect of aTf. Addition to cultures of an antibody against transferrin receptor (TfR), protein kinase inhibitors, or a cyclic AMP (cAMP) analogue showed that a functionally intact TfR was necessary, and that tyrosine kinase, protein kinase C and A, as well as calcium-calmodulin-dependent kinase (Ca-CaMK) activities appeared to mediate aTf actions upon the expression of the above mentioned genes. Changes in the levels of phosphoinositides and cAMP induced by aTf in oligodendroglial cell (OLGc) cultures correlated with these results and coincide with an activation of the cyclic response element binding protein (CREB) and of mitogen activated protein kinases. The increased expression of certain myelin genes produced by aTf appear to be mediated by interaction of this glycoprotein with its receptor, by the cytoskeleton of the OLGc, and by a complex activation of protein kinases which lead to CREB phosphorylation.