The effect of pO2s reduced below physiological levels on GSIR by isolated islets of Langerhans was investigated with a microperifusion apparatus that provided control of pO2 and rapid dynamic response. Second-phase insulin secretion was reduced substantially by hypoxia. The response to lower pO2 was rapid and reversible. Although the steady, normoxic (pO2 = 142 mmHg) second-phase secretion rate varied widely from one islet preparation to another, the ratio of Sx to S142 for each preparation could be represented by a single curve that exhibited a continuous reduction with decreasing pO2. For rat islets perifused 1 day after isolation, the secretion rate was nearly 100% of the normoxic value at a pO2 of 60 mmHg, 50% at 27 mmHg (P50, the pO2 at which the S142 is reduced by 50%), and approximately 2% at 5 mmHg. Oxygen sensitivity of second-phase secretion rate declined after 1 wk of in vitro culture: P50 was 13 mmHg after 1 wk and remained at 10 mmHg after 2-5 wk of culture. Canine islets exhibited a P50 of 16 mmHg after 1 wk of culture. The reduction in insulin secretion is thought to be associated with the existence of pO2 gradients outside and inside the isolated islets, resulting in exposure of islet cells to low pO2 levels that decrease radially from the periphery to the core. We hypothesize that the effect of low pO2 on S is manifested through depletion of the energy stores of the beta-cells. The effect of hypoxia on S may be an important factor in some in vitro secretion studies and may play a critical role in the effectiveness of transplanted islets before their revascularization and of immunoisolated islet implantation devices.