The physiology of gastric epithelial cells is often studied using cancer cell lines, which may, or may not provide information relevant to normal cells. Because few models exist to study chief cell physiology, in vitro, our purpose was to develop primary cultured chief cells from rodent species that are structurally and functionally similar to native chief cells. For this, isolated chief cells from the rat stomach, purified by counterflow elutriation and density gradient centrifugation, were grown in media with growth factors. Purity and the continuity of tight junctions were determined and permeability, viability, TER, cell number and proliferation, and pepsinogen secretion in response to carbachol were measured. When plated in media alone or with bFGF, the isolated chief cells attached by 2 days and were confluent by 4 days after seeding. However, tight junctions were discontinuous, TER was less than 300 Ohm•cm², and permeability was high. In contrast, chief cells incubated with HGF were confluent in 3 days, had a TER greater than 2,000 Ohm•cm², continuous tight junctions, and low permeability. EGF was intermediate. HGF facilitated monolayer development by increasing cell number, which occurred by the proliferation of chief cells. Chief cell cultures, grown with HGF, consisted of more than 99% gastric intrinsic factor-expressing cells and showed robust pepsinogen secretion. Co-expression studies for neck and chief cell markers suggest that the cultures are a mixture of mature, immature, and transitional zone cells. This model will be useful for investigating mechanisms that regulate chief cell physiology in health and disease.