To study the regulation of pepsinogen secretion by chief cells, we have developed techniques for the isolation, enrichment, and short-term culture of chief cells from canine stomach. The fundic mucosa was enzyme dispersed and chief cells were enriched to a content of about 70% using an elutriator rotor. After 36 h in culture confluent monolayers formed that were highly enriched in chief cells. Carbachol induced a time-dependent release of pepsinogen into the medium, with about a threefold increase in pepsinogen secretion over controls found after 60 min of incubation. Carbachol stimulation of pepsinogen secretion was dose dependent, with 5 microM producing 50% of the maximal response found at a carbachol concentration of 100 microM. Atropine (100 microM) produced a rightward shift of the dose-response curve, indicating the presence of a muscarinic receptor. Dibutyryl cAMP, 8-bromo-cAMP, and forskolin also markedly stimulated pepsinogen secretion. Secretin and vasoactive intestinal peptide (VIP) stimulated pepsinogen secretion, but the response were of smaller magnitude than found with carbachol or the cAMP analogues. The phosphodiesterase inhibitor isobutylmethylxanthine also caused a small stimulation of pepsinogen secretion but did not enhance the response to secretin or VIP. These findings indicate that epithelial monolayers can spontaneously form from isolated canine chief cells and retain functional differentiation evident by a response to stimulation. Canine chief cells in culture possess muscarinic and secretin receptors and respond to cAMP.