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 S_x 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 ∼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 β-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.