We have examined the potential roles of intracellular Ca2+ regulation and of multiple cytoskeletal elements in control of the directed migration of cultured oligodendrocyte progenitor cells (OPs). OPs were found to migrate in response to platelet-derived growth factor (PDGF) or to a lesser extent to basic fibroblast growth factor (FGF) in a non-additive manner. This response was inhibited by chelation of intracellular Ca2+ by using BAPTA-AM. OP migration was not evoked by the neurotransmitter agonists phenylephrine or methacholine, which elevate OP Ca2+ levels. Inhibition of the MAP kinase pathway with PD 098059 did not affect OP migration to PDGF. Within growth cone-like leading edges of migratory OP processes, monomeric and filamentous actin were found to be colocalized with myosin and filamentous actin was prominent in filopodia extending beyond the leading edge. Tubulin was distributed throughout OP processes and cell bodies. Inhibition of actin or tubulin polymerization, by using cytochalasin B or nocodazole, respectively, altered OP morphology and markedly impaired migration. Inhibition of the myosin ATPase by BDM, which prevents force-generating actin/myosin interactions, greatly inhibited the chemotaxic response at concentrations that did not disrupt cell morphology. These results indicate that growth factors stimulate OP migration by activating pathways which include intracellular Ca2+ regulation, and characterize the distribution of multiple cytoskeletal elements involved in the generation of directed OP movement.