Oxyrase is an enzyme mixture coveted by microbiologists for its unique ability to remove O2 from media in which anaerobic bacteria are grown. The study reported here examined the potential usefulness of Oxyrase as an adjunct to gassing freshly isolated rat proximal tubules (RPT) with 95% N2-5% CO2 in an attempt to achieve totally O2-free conditions (anoxia) before initiating studies on the mechanism of O2 deprivation injury in vitro. RPT, in 6 ml of Krebs-Henseleit buffer (KHB), were initially gassed with 95% N2-5% CO2 at 1.5 liters/min for 5 min and incubated for 15 to 30 min at 37 degrees C in a shaking water bath, pO2 decreased from approximately 400 to 80 mm Hg. If RPT were present in the KHB, pO2 was even lower, i.e., approximately 50 mm Hg. Addition of increasing concentrations of Oxyrase (300 to 1,500 mU) to KHB alone, that is, without RPT, reduced pO2 from 80 mm Hg to less than 5 mm Hg; increasing the gas rate from 1.5 to 3.0 liter/min of 95% N2-5% CO2, the concentration of Oxyrase to 1,800 mU, and adding RPT reduced pO2 to zero. In this latter condition, pO2 remained unmeasurable during the 20 min of study and neither pH nor pCO2 changed compared with control values. Oxyrase (1,800 mU) had no effect on lactate dehydrogenase release, a sign of membrane injury, in normoxic RPT in KHB. We conclude that anoxia can easily be achieved by the addition of Oxyrase to KHB in which RPT are suspended, if the appropriate concentration of Oxyrase is added and if the RPT are gassed with 95% N2-5% CO2. This concentration of Oxyrase exerts no detrimental effects on RPT gassed with 95% O2-5% CO2.