Few in vitro models expressing complex hepatocyte polarity are available. We used the unpolarized rat Fao cell line to isolate the polarized WIF-B line. These complex rat-human hybrid cells form functional simple bile canaliculi. To obtain Fao-derived polarized models with a simpler chromosome content and developed bile canaliculi, we employed two approaches. Partial success was achieved with monochromosomal hybrids. As shown by the immunolocalization of apical, basolateral, and tight-junctional proteins, monochromosomal hybrid 11-3 cells were polarized. They formed simple functional bile canaliculi and transiently expressed the typical polarity of simple epithelial cells. One subclone blocked in this polarity state was isolated. A more robust approach was provided by spheroid culture, a three-dimensional system that strengthens cell-cell contacts. Transient spheroid culture induced irreversible polarization of Fao cells. This induction occurred in most spheroids (approximately 1% of the cells). From populations enriched in stably polarized cells, we generated new polarized cell models, designated Can. Can 3-1 cells formed simple functional bile canaliculi when plated at high density. Regardless of plating density, Can 9 and Can 10 cells formed long tubular branched canaliculi competent for vectorial transport of organic anions and bile acids, and involving several dozen adjacent cells. Thus, we have generated new cell models stably expressing typical hepatocyte polarity. Among these models, Can 9 and Can 10 are the first capable of forming functional, highly developed bile canaliculi similar to those formed in vivo.