AbstractWe have recently shown that acetylated α‐tubulin containing microtubules (acety1‐MTs; labeled by antibody 6‐11B‐1) constitute a cold‐stable subset of the microtubule network of nonneuronal cells in rat primary forebrain cultures [Cambray‐Deakin and Burgoyne: Cell Motil. 8(3):284–291, 1987b]. In contrast, tyrosinated α‐tubulin containing MTs (tyr‐MTs; labeled by antibody YL1/2) are cold‐labile. Here we have examined the distribution of acety1‐MTs and tyr‐MTs in cultures of newborn rat forebrain astrocytes and simultaneously investigated the distribution of mitochondria and glial filaments. In double‐label immunofluorescence experiments a marked colocalisation of acetyl‐MTs and glial filament bundles was observed. Tyr‐MTs did not show a similar colocalisation with glial filament bundles. Furthermore, the distribution of mitochondria closely followed that of the acetyl‐MT and glial filament bundles. When cells were exposed to short‐term (30‐min) treatments with MT‐disrupting agents such as colchicine and nocodazole, the tyr‐MT network was removed but the distributions of acetyl‐MTs, glial filaments, and mitochondria were unchanged. Increased exposure to colchicine (9–16 hr) caused a progressive disruption of the acetyl‐MTs and the collapse of glial filaments and mitochondria to the perinuclear region. These results suggest that acetyl‐MTs and glial filaments but not tyr‐MTs may be involved in the intracellular transport of organelles and/or in the control of their cytoplasmic distribution.