Rat liver microsomes were found to contain a small pool of nonheme, nonferritin iron. In the presence of ADP, low concentrations of tert-butyl hydroperoxide promoted the reductive release of nonheme, nonferritin iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. Iron release was inhibited by SKF 525-A and metyrapone, which are known to interfere with cytochrome P450-catalyzed reactions. Iron release was also inhibited by high concentrations of t-BOOH, which caused rapid and extensive destruction of cytochrome P450. These observations suggested that iron release was catalyzed by cytochrome P450. Treatment of rats with phenobarbital (PB) caused simultaneous increase of cytochrome P450 and decrease of nonheme, nonferritin iron. The effects of PB were minimized by simultaneous administration of hematin, an inhibitor of heme synthesis, indicating that the nonheme iron was utilized for the synthesis of the heme iron of inducible cytochrome P450 isozymes. Consistently, microsomes from the liver of PB-treated rats were found to release low amounts of Fe2+, unless rats had also been treated with hematin to prevent utilization of nonheme, nonferritin iron for the synthesis of heme iron.