The mechanism by which hormones and neurotransmitters regulate fluid secretion in exocrine glands apparently involves the regulation of transmembrane movements of electrolytes, a process for which Ca serves as a second messenger. Analysis of the kinetics of efflux of 86Rb+ (a marker for K+) indicates that the initial phase of the response to secretagogues is mediated through the release of Ca from a cellular pool inaccessible to chelating agents. By investigating the movements of 45Ca under nearly steady-state conditions, we find that this cellular pool can be filled from the extracellular space without a concomitant elevation in ionized intracellular Ca2+. This suggests that the cellular pool is probably associated with the plasma membrane. We have also investigated the possible role of phosphatidic acid in the mechanism by which receptors mobilize Ca2+. Our results suggest that phosphatidic acid, formed on receptor activation, may directly mediate Ca influx into the acinar cell.