WIF-B cells were generated previously to obtain a goodin vitromodel expressing the structural and functional polarity of hepatocytes. Here we tested the stability and the strength of the WIF-B polarized phenotype. WIF-B cells stayed polarized and formed functional bile canaliculi even after 3 months in culture or after injection in nude mice and culture of the resulting tumors. WIF-B was subcloned and 10,000 colonies were examined; all (except for 3) were composed of bile canaliculi forming cells. Some subclones were characterized; the polarized ones presented the same properties and karyotype as the WIF-B cells; the 3 unpolarized subclones had a lower level of E-cadherin and different karyotypes. WIF-B cells were fused with their nonpolarized hepatic parental cells. The polarity state of the resulting FWIF hybrids was studied from day 11 to day 38 after fusion, by immunolocalization of hepatocyte domain-specific plasma membrane proteins. Most FWIF colonies (>80%) were composed of polarized cells. Soon after fusion these cells were exclusively polarized as simple epithelial cells. The percent of colonies containing cells expressing the typical hepatocyte polarity increased with time and reached 80% at day 38. This result confirms the two-step polarization process previously described for WIF-B. Chromosomally complete FWIF hybrids were examined several months after fusion. As shown by the study of bile acid transport and by confocal analysis of the localization of membrane domain markers, FWIF cells expressed a functional and fully polarized hepatic phenotype. In conclusion, polarity is a stable and dominant trait of WIF-B.