Complete disruption of the microtubular network by colchicine or nocodazole in Caco-2 intestinal epithelial cells results in the appearance of basolateral microvilli and brush border-containing intracellular vacuoles (vacuolar apical compartment: VAC). These vacuoles are surrounded by a terminal web, express apical markers and exclude basolateral markers. The vacuoles do not originate from internalized apical or basolateral plasma membrane and their development is blocked by protein synthesis inhibitors, suggesting that they are newly synthesized. After removal of the microtubule inhibitors, VACs are usually degraded and/or released into the lateral intercellular space. Rarely was fusion with the apical membrane observed. These experiments support a role for microtubules in the biogenesis of the apical surface and indicate that, under some conditions, apical plasma membrane assembly may occur in the cytoplasm, as observed in some human pathological states.