Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors (in the presence of serum) resulted in loss of polarity although the cells retained growth responses to both factors. Coculture of BECs with autologous human hepatocytes enhanced the ability of low-density BECs to undergo ductulogenesis. This effect was mimicked by addition of conditioned medium from previous hepatocyte-BEC cocultures. These findings indicate that for human biliary ductular morphogenesis, epithelial cell-cell interactions are required but that mesenchymally derived factors such as HGF may not be important.