Fasting for 24 or 72 h causes a strong decrease of pancreatic islet calcium content as detected by glyoxal-bis-(2-hydroxyanil), (GBHA). There is strong evidence that GBHA only detects ionized calcium and not total calcium (Wolters et al., 1979). Fasting does not influence the zinc content as detected by dithizone (DZN), and aldehyde-fuchsin (AF) staining intensity is only slightly decreased. After degranulation of islets by tolbutamide (which reduced the insulin content of the pancreas to 10% of the control value) the staining intensities of GBHA, DZN and AF were strongly depressed. Calcium (as well as other elements) were also measured by electron-probe micro-analysis (EPMA). It appeared that 24 or 72 h of fasting did neither affect the total content of Ca nor of Na, P, S, and K of the islets significantly. In exocrine tissue the Ca content increased gradually as a result of fasting. Thus, after 72 h of fasting the Ca content was 25% higher than in fed controls. On the other hand after 72 h of fasting the K content appeared to be decreased. EPMA revealed that after degranulation of islets the Ca content decreased markedly (35%). S appeared to be decreased by only 14%, whereas the content of the other elements was not changed. The results show that GBHA-detectable Ca is only a part of EPMA-detectable Ca. The GBHA-Ca "pool" which contains ionized Ca, is subjected to changes when the animals are fasted, the total Ca content as measured by EPMA does not change. Thus, at least two distinguishable pools of Ca exist within the islets (GBHA-detectable and not-GBHA-detectable). It is suggested that as a result of fasting Ca passes from one pool to another.