Aminopyrine N-demethylase was inhibited by phenelzine in vitro and in microsomes isolated from rats treated with phenelzine in vivo. The inhibition was greater if phenelzine was incubated with the microsomal suspension before the addition of the substrate. In vitro, the addition of phenelzine produced immediate decrease in cytochrome P-450. This initial decrease was found to be due to the direct binding of phenelzine to the ferrous heme of cytochrome P-450. Further decrease in cytochrome P-450 occurred upon incubation of microsomes with phenelzine in the presence of NADPH. This secondary decrease in cytochrome P-450 was found to be paralleled by a loss of the home content in cytochrome P-450. The decrease in cytochrome P-450 with concomitant loss of heme can be partially inhibited by a substrate (aminopyrine) or by an inhibitor (metyrapone) of the microsomal enzymes, indicating the possible involvement of the metabolism of phenelzine to a reactive intermediate. The comparison of the effect of phenelzine to that of phenylhydrazine strengthened the possibility that a reactive metabolic intermediate of phenelzine can cause heme destruction in cytochrome P-450. Thus, phenelzine exerts its inhibitory action on microsomal drug metabolism most likely by decreasing the cytochrome P-450 content largely through heme destruction.