The intravenous C-erythromycin breath test (EBMT) has been largely used in adults as a validated probe to measure hepatic cytochrome P450 3A4 and 3A5 (CYP3A4/5) activity in vivo. Additionally, the oral EBMT has been suggested to measure combined hepatic and intestinal CYP3A4/5 activity. Both hepatic and intestinal CYP3A4/5 activities are low in neonates, but the exact developmental pattern is not known. Also, a knowledge gap exists on the impact of comedication or disease state on CYP3A4/5 activity in this population. However, to use the radioactive test in newborns is not feasible, for obvious ethical reasons. Hence, the aim of this pilot study was to determine if stable isotope-labeled C-erythromycin could be used alternatively. Preterm infants who needed treatment with erythromycin for ureaplasma infection were given an oral 10 to 15 mg/kg C-(N-dimethyl)-erythromycin dose. Pharmacy regulations did not permit intravenous administration. Exhaled air samples were collected predose and up to 24 hours post-dose and analyzed for CO2 and CO2 with gas chromatography-mass spectrometry. Three patients received oral C-erythromycin. CO2 did not change significantly from baseline, showed a maximum blood concentration at 20 hours (+12%), and decreased over 24 hours (-16%) in these patients, respectively. Because none of these patients showed a consistent peak in C enrichment, in accordance with maximum blood concentration of oral erythromycin in preterms, we stopped this pilot trial after 3 patients. In conclusion, the lack of a consistent change in exhaled CO2 after oral C-erythromycin in this pilot study precludes the routine use of oral 13C-EBMT in preterm infants as a noninvasive probe of CYP3A4/5 activity. We speculate that this lack of change is due to developmentally low intestinal and hepatic CYP3A activity.