Studies on hormone action in isolated islets have generally been carried out using concentrations far above physiologic levels. This study investigates whether glucagon at concentrations close to the physiologic level is insulinotropic in isolated islets and how this relates to islet cyclic AMP levels. Collagenase isolated rat islets were tested directly after isolation or after a 24-hour tissue culture. Insulin release and islet cyclic AMP content were determined during a 30-minute incubation by radioimmunoassay. After maintenance in tissue culture glucose-induced (16.7 mmol/L) insulin release was clearly enhanced by glucagon concentrations between 2 and 1,000 ng/mL in a dose-related manner. Islet cyclic AMP was increased only by glucagon 1 mumol/L (3.8 micrograms/mL). When phosphodiesterases were inhibited (0.1 mmol/L 3-isobutyl-1-methylxanthine) insulin release was stimulated by 1 ng/mL and cyclic AMP by 100 ng/mL. By contrast, in freshly isolated islets, glucagon concentrations in the range of 1 to 100 micrograms/mL were needed to augment glucose-induced insulin release. These findings demonstrate that the hormone sensitivity of collagenase isolated islets is markedly improved by short-term maintenance in tissue culture. The threshold level for a detectable effect on islet cyclic AMP is considerably higher than for glucose-induced insulin release. Since in hepatocytes two signal transduction systems for glucagon have recently been demonstrated, the results could mean that at low concentrations glucagon effects may not be mediated via cyclic AMP.