Microtubules 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.