AbstractMicrotubules oriented in the apicobasal axis of columnar epithelial cells arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF‐B (hepatocyte), Caco‐2 (intestine), and Madin‐Darby canine kidney (MDCK). Caco‐2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole‐induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF‐B and Caco‐2 cells contained two pools of γ‐tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non‐centrosomal γ‐tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal γ‐tubulin in apical cytoplasm suggests multiple mechanisms by which microtubule nucleation might occur in epithelial cells. © 1995 Wiley‐Liss, Inc.