The metabolism of N,N-dimethylaniline (DMA) by rabbit liver microsomes results in the formation of N-methylaniline (NMA) and formaldehyde. The N-oxide of DMA (DMA N-oxide) has been suggested as an intermediate in the cytochrome P-450-catalyzed demethylation reaction. The role of DMA N-oxide as an intermediate in demethylation has been investigated in a reconstituted system consisting of NADPH-cytochrome P-450 reductase, phospholipid, and several different purified isozymes of cytochrome P-450. The abilities of several cytochrome P-450 isozymes from rabbit liver (P-450 form 2 and P-450 form 4) and rat liver (P-450b and P-450c) to catalyze N-oxide formation and their abilities to catalyze demethylation of the N-oxide were determined and compared with their abilities to catalyze the demethylation of DMA. The metabolism of DMA by the purified isozymes of cytochrome P-450 in the reconstituted system did not result in the formation of measurable amounts of the N-oxide. The turnover numbers for the metabolism of DMA and DMA N-oxide to formaldehyde by the reconstituted system containing cytochrome P-450 form 2 were 25.6 and 3.4 nmol/min/nmol cytochrome P-450, respectively. The three other isozymes (P-450 form 4, P-450b, and P-450c) also exhibited significantly greater rates for the demethylation of DMA than for the N-oxide. If the N-oxide were an intermediate in the demethylation reaction, it should be metabolized at a rate greater than or at least equal to DMA. Therefore, these data, along with the inability to detect N-oxide formation during the cytochrome P-450-catalyzed demethylation of DMA, suggest that the N-oxide of DMA is not an intermediate in demethylation of DMA by these forms of cytochrome P-450 and that DMA N-oxidase activity is not associated with these isozymes.