It is generally accepted that process-bearing microglial cells origina te from ameboid macrophage-like mesodermal cells. This transformation, often called ramification, accompanies down regulation of macrophage-like properties, but the mechanisms involved in ramification have not been clarified. We investigated factors to promote ramification in culture. Isolated ameboid microglial cells were seeded on living or paraformaldehyde-fixed astrocyte monolayers. About 80% of the cells ramified on the fixed astrocytes in astrocyte-conditioned medium as well as on the living astrocytes. In fresh culture medium, 50% of the cells on the fixed astrocytes ramified. On the other hand, ameboid cells rarely ramified on noncoated glass coverslips even in the conditioned medium. Ameboid cells cultured on extracellular matrices dervied from astrocytes ramified more than on those coated with plasma fibronectin or collagen type I. A synthetic peptide containing Arg-Gly-Asp sequence or a tyrosine kinase inhibitor genistein partially reversed the ramification induced on the fixed astrocyte monolayers. These results show that some nondiffusible factors derived from astrocytes are essential for microglial ramification. A part of the nondiffusible factors are present in the extracellular matrices, and the effects might be mediated by integrins. Some diffusible factors secreted by astrocytes seem to promote ramification, if the nondiffusible factors are present. The experiments using the fixed astrocyte monolayers may be useful to identify the diffusible factors responsibile for ramification.