In the present study we examined the actions of various secretagogues on pepsinogen secretion from freshly dispersed chief cells prepared from guinea pig stomach. Chief cells were obtained by preparing dispersed gastric glands, subjecting the glands to mechanical disruption in the presence of EGTA, and fractionating the resulting mucosal cells on a Percoll density gradient. Chief cells constituted 90% of the final cell suspension and cell viability was 99%. In these cells, pepsinogen secretion was stimulated by agents whose actions are probably mediated by calcium: carbachol, cholecystokinin, and A23187. Pepsinogen secretion was also stimulated by agents whose actions are probably mediated by cAMP: secretin, vasoactive intestinal peptide, and 8-bromo-cAMP. Reducing the incubation temperature from 37 degrees to 4 degrees C or adding carbonyl cyanide m-chlorophenylhydrazone abolished secretagogue-induced pepsinogen secretion. These results indicate that freshly dispersed chief cells from guinea pig stomach are responsive to secretagogues and provide a suitable model for investigating cellular mechanisms of secretagogue-induced pepsinogen secretion.