Because there is considerable interindividual variation in both microsomal CYP3A4 activity and CYP3A4 substrate disposition, an established probe of in vivo CYP3A4 activity would represent an important advance in clinical practice. In a previous study, no correlation was found between the 14C-erythromycin breath test and urinary dapsone recovery ratio. However these drugs were administered by different routes, with the orally administered dapsone being exposed to presystemic metabolism by the gut and renal metabolism before the measurement of the urinary ratio. To overcome the variable of route of administration, the aim of this study was to determine whether the elimination of two intravenously administered CYP3A4 substrates (alfentanil and erythromycin) correlate. We compared the metabolism of alfentanil to its CYP3A4-dependent metabolite, noralfentanil, with the erythromycin breath test in 14 young healthy white men. No significant correlation was found between alfentanil metabolism and the erythromycin breath test: alfentanil clearance versus erythromycin breath test, r = 0.45, p = 0.1; partial metabolic clearance to noralfentanil versus erythromycin breath test, r = 0.35, p = 0.23. Because these two CYP3A4 substrates were administered by the same (intravenous) route, we conclude that differences in the route of administration do not explain the lack of correlation between the erythromycin breath test and other probes of CYP3A4 metabolism.