1. A study has been made of the calcium movements in isolated acinar cells of rabbit pancreas in relation to the process of enzyme secretion. 2. After 90 min, the 45Ca2+ uptake level of the acinar cells reaches a steady state level which depends on the extracellular calcium concentration: it increases with increasing calcium concentration. 3. Carbachol, in addition to stimulating enzyme secretion, causes a decrease in the 45Ca2+ content of pre-loaded acinar cells. This decrease is virtually independent of the extracellular calcium concentration. It is followed by an uptake of 45Ca2+ which only in a medium with 2.5 mM Ca2+ leads to a significantly higher 45Ca2+ level than the steady state level. 4. Carbachol, added to acinar cells not preloaded with 45Ca2+, increases the 45Ca2+ uptake in medium with 2.5 mM and in that with 0.1 mM Ca2+. 5. The amount of enzyme released by carbachol depends on the extracellular calcium concentration: it is larger in media with higher calcium concentration. 6. It is concluded that the increase in cytoplasmic calcium concentration which takes place immediately upon addition of a stimulus and which is necessary for the stimulation of the enzyme secretion, is caused by a release of calcium from an intracellular pool and not by an influx of calcium from the extracellular medium. 7. The results suggest that there are at least three different calcium pools in the pancreatic acinar cells.