Isolated cells from the nasal salt gland of ducklings (Anas platyrhynchos) were evaluated as a model system for the study of the muscarinic activation of exocrine ion secretion. Cells loaded with the fluorescent probe indo-1 were used to study changes in intracellular Ca2+ concentration [( Ca2+]i) after stimulation. Changes in inositol phosphate generation and oxygen consumption were also determined. Loading with the acetomethoxy ester form of indo-1 (indo-1/AM) was rapid, and intracellular cleavage of the ester was essentially complete. Leakage of the dye was negligible over the time course of measurements (up to 20 min). Resting [Ca2+]i was approximately 100 nM. Stimulation with carbachol resulted in progressive increases in the generation of inositol phosphates and rapid four- to fivefold increases in [Ca2+]i. At normal extracellular Ca2+ concentrations, [Ca2+]i remained elevated (approximately 3 times resting levels) for as long as stimulation continued. Experiments showed that the increases in [Ca2+]i were comprised of a combination of release of Ca2+ from intracellular stores and an enhanced entry of Ca2+ from the extracellular medium. It is specifically this latter process that produces the sustained elevations in [Ca2+]i that are the essential signal for secretory activity.