Various forms of stress result in decreased O2 permeability or decreased capacity to consume O2 in legume root nodules. These changes alter the nodule interior O2 concentration (Oi). To determine the relationship between Oi and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled Oi by varying external pO2 while monitoring internal H2 concentration (Hi) with microelectrodes. Oi was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in Oi, Hi approached a new steady state, with a time constant averaging 23 s. The rate of H2 production by nitrogenase was calculated as the product of Hi, nodule surface area, and nodule H2 permeability. H2 permeability was estimated from O2 permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. Oi was nearly optimal for nitrogenase activity (H2 production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO2 (40-100 kPa) reduced Hi to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO2. The mechanism of nitrogenase inhibition may differ between brief and prolonged O2 exposures.