(1) In order to determine the cellular localization of the secretin- and pancreozymin-sensitive adenylate cyclase in rat pancreas, the occurence of this enzyme system has been investigated in isolated pancreatic cells. (2) Digestion of rat pancreatic lobules with collagenase yields a preparation of isolated cells which upon differential morphological analysis appears to consist for 97% of acinar cells and to contain for fewer centro-acinar and ductal cells than undissociated lobules. (3) Expressed per mg protein, the isolated cells contain the same amount of DNA, chymotrypsin and lactic dehydrogenase as the undissociated tissue. The stimulated adenylate cyclase activity is nearly entirely recovered in the isolated acinar cells, as is also the case for the low Km adenosine 3',5-cyclic monophosphate phosphodiesterase activity and the adenosine 3',5'-cyclic monophosphate (cyclic AMP) content. Marked losses are noted for the basal adenylate cyclase and the high Km cyclic AMP phosphodiesterase activities. (4) Washing the isolated acinar cells in Krebs-Ringer bicarbonate medium containing 10 mM 1-methyl-3-isobutylxanthine causes a cyclic AMP level 2.6 times that in cells washed in Krebs-Ringer bicarbonate alone. The cyclic AMP level is further increased by subsequently incubating the cells for 10 min in the presence of 3-10(-7) M pancreozymin-C-octapeptide or secretin to values 1.7 or 4.7 times the control level in cells incubated for 10 min with 1-methyl-3-isobutylxanthine alone. (5) It is suggested that the adenylate cyclase of the acinar cells may be involved, with another factor, in the stimulation of enzyme secretion, whereas a ductular cyclase would function in the regulation of the bicarbonate-dependent fluid secretion.