To study the fate of volatile fatty acids (VFA) in the large bowel, we compared the rate of oxidation of 13C-labeled VFA administered rectally with that of the orally administered substrate. On two different days, 1-[13C]acetate was administered rectally or orally to five infants recovering from diarrhea. Breath samples were collected over 4 h and analyzed for 13C enrichment of breath CO2 by gas isotope ratio mass spectrometry. The percent dose recoveries of 13C in breath were fitted to multicompartmental models using the SAAM-27 program. Following model development procedures, the oral acetate breath test curves could be accounted for only by a compartmental model in which labeled acetate underwent absorption into and mixed with a systemic pool before oxidation took place. The rectal acetate breath test curves could be accounted for by a simpler model in which oxidation occurred directly in the compartment in which the rectal acetate was administered, and required no rate-limiting absorptive process. Our results indicate that the labeled acetate was oxidized more rapidly when the substrate was administered rectally than orally. This observation points to the direct utilization of volatile fatty acids within the colon.