A novel method for generating nontransformed human intestinal primary epithelial cell (HIPEC) lines in an in vitro culture system is reported here. Although several groups have reported the development of nontransformed intestinal epithelial cells (IEC) lines (Deveney et al, 1996; Latella et al, 1996; Pang et al, 1996; Perreault and Beaulieu, 1998), it still had been difficult to find an optimal condition to generate a pure population of nontransformed IEC in long-term cultures. It was hypothesized that an appropriate growth factor/cytokine milieu that would mimic the physiological microenvironment might favor the survival of the isolated cells and might play a critical role in epithelial cell growth. To test this hypothesis, isolated progenitor/crypt cells were cultured in collagen-coated petri dishes in the presence of mucosal tissue-derived growth factor containing culture supernatants (14-18 hours) and a combination of hormonal supplements. Cell attachment and growth was observed within 24 hours and confluent monolayers were seen between 7 and 12 days. Immunofluorescence staining and flow cytometric analysis of the cells demonstrated positive staining with anti-cytokeratin-18 antibody confirming their epithelial origin. The reproducibility of the method has been confirmed by establishing a number of HIPEC lines from various segments of the gastrointestinal tract. This novel method of HIPEC line generation, which maximizes the similarity of the ex vivo culture system to in vivo conditions, will serve as a valuable tool for the establishment of a large number of HIPEC lines (intestinal epithelial cell bank) and for subsequent use in studies of the immunological/physiological epithelial function in the intestine.