Junctional adhesion molecule, JAM, is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knock-down (KD) and over-expression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A shRNA resulted in ~~50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When RNAi resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ binding motif (huC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Over-expression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that: JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins; and hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.