Little is known about the mechanisms that establish and maintain the proliferation and differentiation programs of the gastric epithelium. This is largely due to the complexity of the gastric epithelial units and the presence of the different epithelial lineage progenitors among heterogeneous populations of various mature cell types. This study is undertaken to establish an in vitro system highly enriched for gastric epithelial lineage progenitors. By using adult male rabbits, a simple method of isolating gastric epithelial cell fractions enriched in lineage progenitors was applied. Cultured cells labeled with bromodeoxyuridine were characterized by using lectin and immunohistochemical studies at light- and electronmicroscopical levels. Analysis of primary cultures derived from the progenitor cell region of the epithelial units revealed that this system can support the proliferation and some of the differentiation programs of the progenitor cells. Cultured cells can be maintained for up to 5 days, while retaining most of the morphological features, molecular markers, and dynamic behavior of gastric epithelial progenitors. Differential cell counts at 1-day culture revealed that, while the undifferentiated progenitors formed about 30% of all attached cells, pre-pit, pit, and preparietal cells represented about 30%, 10%, and 2%, respectively. By 3 days, the increase in the percentage of pit and preparietal cells up to 25% and 9%, respectively, reflected their production in vitro. In conclusion, we have established a culture system enriched for gastric epithelial lineage progenitors that would hopefully allow the identification of factors and mechanisms involved in controlling their proliferative activity and differentiation pathways.