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The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms.