Colony-stimulating factors are important mediators of microglial growth and function. Microglia, derived from ameboid precursor cells, undergo a series of morphological changes in the central nervous system to become ramified, a morphological hallmark of differentiated microglia. The cellular mechanisms and environmental factors involved in microglial ramification in the developing brain are not known. In the present study, we examined the role of macrophage colony-stimulating factor (M-CSF) in the induction of ramification of microglia in human fetal glial cultures. Human fetal microglia underwent spontaneous conversion from ameboid to ramified shape when co-cultured with human fetal astrocytes. Concurrently, high levels of M-CSF also accumulated in cultures of unstimulated human fetal astrocytes. That the M-CSF was essential in inducing microglial ramification was demonstrated by almost complete inhibition of ramification by an antibody to human M-CSF receptor, c-fms. Furthermore, profound inhibition of microglial ramification was exerted by herbimycin A, a specific inhibitor of tyrosine kinase (a component of the signal cascade of c-fms), supporting the role of M-CSF/c-fms in the induction of microglial ramification. Our results suggest a pivotal role played by astrocytes and astrocyte-produced M-CSF in the induction of microglial maturation and differentiation in the developing human brain.