Carbamylcholine (30 μM) increased the concentration of guanosine 3′,5′-monophosphate (cyclic GMP) in guinea pig pancreatic lobules about eight- to tenfold over the basal concentration in 30 seconds with a concomitant increase in the rate of amylase secretion. The concentration of cyclic GMP rapidly declined to a plateau value of about 16 percent of the peak level in 10 minutes. Cellular cyclic GMP decreased, mostly because the nucleotide was secreted into the medium; cellular adenosine 3′,5′-monophosphate (cyclic AMP), however, did not change, nor was this nucleotide secreted into the medium. An immunocytochemical technique showed that cyclic GMP was distributed in the apical plasmalemma membrane and lumen of the pancreas. Carbamylcholine increased the cyclic GMP fluorescence in the apical plasmalemma membrane within 30 seconds, and in zymogen granules and the plasma membrane in the apical part of acinar cells in 10 minutes. The islets of Langerhans did not show any change in cyclic GMP. Fluorescence of cyclic AMP in pancreatic lobules was not altered by carbamylcholine and was localized along the apical portion of plasmalemma and cytoplasm. Cyclic GMP may thus participate either in the process of exocytosis or in the activation of enzymes secreted from the pancreas.