In situ and in vitro, microglia can have different morphologies, which are thought to reflect distinct physiological activities. Two extreme forms are ameboid and ramified microglia. To study cytoskeletal changes during differentiation, we used defined cell culture systems to yield cultures of ameboid or ramified microglia from mouse brain. With respect to proliferation, secretion, receptor-expression, and phagocytosis, ramified microglia was generally less active. We found that the transition to a ramified phenotype was accompanied by an increase in the relative amount of acetylated and detyrosinated tubulin. Whereas the modified microtubules were restricted to regions close to the microtubule-organizing centers (MTOCs) in ameboid cells, acetylated microtubules were abundant in ramified cells, where they appeared to traverse from one process to another with no apparent anchoring at MTOCs. The increase in acetylated and detyrosinated microtubules was paralleled by an increased stability against nocodazole-induced microtubule disassembly and by a lower rate of change in the length of the processes. Staining of retinal wholemounts confirmed the presence of acetylated microtubules in ramified microglia in situ. We conclude that during the transition of microglia to a ramified phenotype regulated processes exist, which cause a reorganization of microtubules and a change in composition of the microtubule skeleton resulting in a less dynamic and more stable microtubule network. Intracellular factors that are specifically involved in microtubule stabilization in ramified microglia need to be identified in future research and may provide a useful criterion for defining ramified microglia.