The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag-period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta-cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion.