This study demonstrates interplay among human hepatic cytochrome P450 (CYP) isoforms in transforming aromatic compounds from being prosubstrates of CYP3A4 into phenolic substrates. Incubation of methoxychlor with CYP2C19 yields the phenolic monodemethylated derivative (mono-OH-M). Additionally, CYP2C19 catalyzes the ortho-hydroxylation of mono-OH-M and of residual methoxychlor. CYP3A4 does not catalyze the O-demethylation or hydroxylation of methoxychlor, but does hydroxylate mono-OH-M (ortho to the phenolic hydroxyl) (Stresser DM and Kupfer D, Biochemistry 36: 2203-2210, 1997). A combination of reconstituted CYP2C19 and 3A4 in the same vessel elicits stimulation of the ortho-hydroxylation of mono-OH-M compared with 2C19 alone. It is unlikely that stimulation of hydroxylation was due to protein-protein interactions, generating more active P450(s), because progression of the stimulation was time-dependent. When reconstituted CYP3A4 was added to an ongoing incubation containing reconstituted 2C19, stimulation of catechol formation occurred. In another experiment, stimulatory activity was similar when 2C19 and 3A4 were reconstituted together in the same vesicles or separately. Cumulative evidence demonstrates that the stimulation of catechol formation resulted from CYP3A4-mediated ortho-hydroxylation of the phenolic metabolite(s) generated by CYP2C19. Similarly, estradiol 3-methyl ether is demethylated by CYP2C19 into estradiol, a CYP3A4 substrate for ortho-hydroxylation; there was significant stimulation of hydroxylation by combined 2C19 and 3A4. These findings demonstrate that pro-phenolic compounds (methoxychlor and estradiol 3-methyl ether) are prosubstrates of CYP3A4. Because catalysis may become evident only after prosubstrate conversion (by a different P450) into a substrate, caution is warranted when concluding a lack of catalytic involvement by a particular P450 isoform, based solely on data from the use of individual cDNA-expressed P450s.