Cellular levels of cAMP and cGMP were measured in guinea pig pancreatic lobules incubated in vitro, during basal or stimulated secretion. Stimulation with optimal concentrations of carbamylcholine (carbachol) (10(-5) M), pancreozymin (0.1 unit/ml), and caerulein (10(-9) M) resulted within seconds in a sharp rise in cGMP levels, from five to more than 20 times that of basal levels. cAMP levels did not change significantly. cGMP increases were maximal at 2 min then subsided by 4-7 min to a plateau about two to three times that of basal level. This plateau was maintained for the duration of the secretagogue stimulus. Removal of the carbachol stimulus resulted in a rapid decrease in cGMP levels to that of the basal state. The cellular cGMP levels observed within the first 2 min of stimulation correlated closely with the dose of carbachol and the secretory response. Atropine at 10(-4) M blocked the cGMP elevation due to carbachol but not that due to pancreozymin, while carbonyl cyanide m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked the response to both secretagogues. Similar though less extensive findings were observed using rabbit pancreatic lobules incubated in vitro. High concentrations (10(-2)-10(-3) M) of the dibutyryl and 8-bromo analogues of both nucleotides were effective, though suboptimal, secretagogues. In the case of the cAMP analogues, the secretory response was associated with a rise in endogenous cGMP levels, similar to that observed during suboptimal carbachol stimulation. These findings suggest that cGMP may be an intracellular mediator in the process of stimulus secretion coupling in the acinar cell of the exocrine pancreas.