Biochemical and biotechnological aspects of aeration conditions in iron- and sulfur-oxidizing Acidithiobacillus ferrooxidans cultures were studied. The critical values of volumetric oxygen transfer coefficient, (kLa)crit, and oxygen concentration, Ccrit, were used to define the conditions necessary for minimum aeration and to eliminate potential oxygen limitation during substrate oxidation by themodel bioleaching culture. The Michaelis constants for oxygenwere 1.07 and 0.61 umol O2/l for the oxidation of ferrous iron and elemental sulfur, respectively. The Ccrit values, below which oxygen limitation may occur, were 6.250 and 3.125 umol O2/l, determined in respirometric experiments for the oxidation of ferrous iron and elemental sulfur, respectively. The rates of iron and sulfur oxidation were also determined at the Ccrit values by measuring the formation of ferric iron and protons in the corresponding cell suspensions. Neither iron nor sulfur oxidation was limited by oxygen at Ccrit. The (kLa)crit values required to maintain oxygen-unlimited oxidation of ferrous iron and elemental sulfur were 7.70 and 4.88/h, respectively.