1. Calcium and magnesium movements in the isolated rabbit pancreas and in rabbit pancreas fragments are compared in a qualitative and quantitative way. 2. At the basal secretion rate calcium and magnesium are present in the secreted fluid in concentrations of about 30% of their concentrations in the bathing medium. 3. Addition of 10(-6) M carbachol to the bathing medium results in enzyme secretion accompanied by calcium and magnesium release, in divalent cation-free medium as well as in a complete medium. 4. The secretion of each divalent cation is the sum of two components: an extracellular flux and a flux of protein-associated cations, the so-called secretory flux. 5. The extracellular flux is proportional to the concentration of the divalent cation in the bathing medium. The secretory flux is not dependent on the presence of the divalent cation in the bathing medium, but is proportional to the amount of protein secreted. About 25 nmol of each cation is secreted per mg protein. 6. Ca2+ and Mg2+ can be nearly completely separated from the digestive enzymes by gel filtration. They equilibrate completely with their radioactive isotopes added to the sample just before elution, indicating that the cations are rapidly exchangeable after secretion. 7. Efflux studies on rabbit pancreas fragments, pre-loaded with 45Ca2+, show a carbachol-stimulated 45Ca2+ efflux (the stimulatory flux) in addition to a release of amylase. Fragments pre-loaded with 28Mg2+ do not show carbachol stimulation of the tracer efflux. 8. These studies indicate that calcium and magnesium behave quite similarly with respect to the extracellular and secretory fluxes. The absence of a stimulatory flux for magnesium, suggests that the increase of the cytoplasmic calcium concentration plays a specific role in the stimulus-secretion coupling of pancreatic enzyme secretion.