Cells were isolated from approximately 30 d fetal rabbit stomachs and cultured on floating collagen gels. Electron microscopy showed monolayers in which only one cell type persisted. These columnar cells were joined at apical borders by tight junctions and contained an extensive endoplasmic reticular network with an occasional intracellular canaliculus. They also occasionally contained what appeared to be secretory granules (mucus?), and therefore had some characteristics of all the cell types of the intact fetal stomachs, which showed oxyntic, mucous, and undifferentiated cells. In Ussing chambers with Ringer's solution on both sides, cultures developed transepithelial potential (potential difference [PD], mV, mucosa ground) = 13, resistance (resistance [R], omega - cm2) - 285, and short-circuit current (Isc, microA/cm3) = 45 (n = 7), clearly indicating that cellular polarity and junctional integrity were maintained. These transport parameters were somewhat different for intact fetal stomachs (PD = 20, R = 70, and Isc = 220 [n = 4]), which may be due to extensive folding of intact fetal stomachs or the presence of only one cell type in culture, or both. Although gastric stimulants histamine, dibutyryl cycle AMP (dbcAMP), and isobutylmethylxanthine (IMX) (a phosphodiesterase inhibitor) did not elicit H+ secretion or electrophysiological changes in monolayers or intact stomachs, 10-4 M apical amiloride caused a decrease in Isc in cultured monolayers(27%) and intact stomachs (50%). Thus, Na+ transport seems to be a significant fraction of ion transport in both preparations. This culture system may allow the study of oxyntic cell differentiation and the development of H+, Na+, and Cl- transport in the gastric musoca.