There are significant interpatient differences in the activity of a major drug metabolizing enzyme termed P450IIIA. Because P450IIIA uniquely catalyzes the N-demethylation of erythromycin, we have proposed that the P450IIIA activity of a patient may be determined from the rate of 14CO2 production in the breath after an intravenous infusion of a test dose of [14C-N-methyl]erythromycin. However, direct evidence that this erythromycin breath test selectively measures P450IIIA and not other major human liver P450s in patients has been lacking. We therefore administered the erythromycin breath test to nine patients with severe liver disease who were awaiting liver transplantation. Microsomes were prepared from liver samples obtained during surgery and the concentration of P450IA2, P450IIC8, P450IIC9, P450IIE1, and P450IIIA were determined immunochemically. We found a significant correlation between patients' erythromycin breath test results and their liver P450IIIA levels (r2 = 0.56, p = 0.02). In contrast, there was no correlation at all between the erythromycin breath test result and the microsomal levels of any of the other four P450s assayed. The correlation of the erythromycin breath test and P450IIIA did not appear related to the extent of liver disease because neither correlated with prothrombin time or albumin or bilirubin levels. These data provide the best evidence to date that the erythromycin breath test is a specific assay of in vivo P450IIIA activity in patients.