A mitochondrial preparation from duck adrenal gland was used, under aerobic conditions, to show that the oxygen requirement for the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) is at the cytochrome P-450 level only. Vitamin C and tetramethyl-p-phenylene-diamine (TMPD) were used to increase oxygen consumption at the cytochrome a3 level, thereby decreasing its availability to cytochrome P-450. The vitamin C plus TMPD system acts as an 'oxygen trap'. Results show that despite reducing equivalents provided by L-malate, vitamin C plus TMPD strongly inhibits aldosterone biosynthesis from 18-hydroxycorticosterone (89%). Moreover, we used KCN in order to block oxygen consumption, even in the presence of vitamin C plus TMPD. Under these conditions, the inhibition of aldosterone biosynthesis from 18-hydroxycorticosterone is reduced by 51%. The reversal of this inhibition by KCN was evident but only partial. According to polarographic and electron microscopy studies, the reversal of inhibition can only be explained by an increased availability of oxygen at the cytochrome P-450 level. Experiments performed under aerobic conditions, without a nitrogen atmosphere, show that oxygen is required in the transformation of 18-hydroxycorticosterone into aldosterone, at the cytochrome P-450 level. This suggests that a classical hydroxylating mechanism is involved.