We have studied pepsinogen synthesis and secretion in primary monolayer cultures of canine gastric chief cells. Monolayers were formed after approximately 48 hr. Pepsinogen synthesis was studied by adding 14C-labeled amino acids to the culture medium. Basal secretion of de novo synthesized pepsinogen after 4 hr was 4 +/- 1.2% of total newly synthesized pepsinogen. Basal secretion of stored pepsinogen after 90 min was 8 +/- 1.4% of total pepsinogen content. Carbachol, dbcAMP, forskolin, VIP, CCK-8, and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate all stimulated secretion of de novo synthesized pepsinogen and preformed pepsinogen. Only additive interactions were found. dbcAMP caused a peak outburst of stored pepsinogen in the first 10 min. Carbachol stimulation was time dependent. Stimulation of de novo synthesized pepsinogen secretion was time dependent for both carbachol and dbcAMP. dbcAMP caused an immediate 10-fold increase in pepsinogen synthesis, but carbachol did so only after a lag time of 30 min. This was identical to the time necessary for the appearance of labeled pepsinogen in the medium. Addition of atropine after 2 hr resulted in a return to basal synthesis. Stimulated pepsinogen synthesis was always observed concomitant with stimulated pepsinogen secretion. These results show that most external stimuli for pepsinogen synthesis are dependent upon prior depletion of pepsinogen stores, which then triggers synthesis, while stimulation of cAMP production stimulates pepsinogen synthesis more directly.