Many microorganisms in the large intestine are capable of synthesizing folate. Preliminary evidence suggests that this folate may be absorbed. The purpose of the 2 experiments reported herein was to estimate the pool of folate in the feces of human infants and piglets and to ascertain, if absorbed, whether the quantity and form of folate are sufficient to potentially affect the folate status of the host organism. The folate content of milk fed to and of fecal solids collected from exclusively human milk-fed (n = 12) and formula-fed (n = 10) term infants (1-6 mo old) was determined microbiologically before (short-chain folates) and after folate conjugase (total folate) treatment. The folate content of formula fed and of feces collected from 10-d-old piglets (n = 10) was also determined microbiologically. The proportion of 5-methyltetrahydrofolate (5-methylTHF) in feces of human infants and piglets that was monoglutamylated was determined by HPLC analysis. The folate content of fecal solids collected from infants was 93.2 ± 92.8 nmol/d (mean ± SD), representing on average 50% (8.0-170.1%) of their mean estimated dietary folate intake. Fecal folate was largely present as short-chain folate (66 ± 21.3%) with the predominant form being 5-methylTHF, 52.5 ± 30.1% of which was monoglutamylated. In piglets, the folate content of feces was 301.3 ± 145.7 nmol/d, representing 36% of their dietary folate intake. Piglet fecal folate was largely present as short-chain folate (68.1 ± 12.6%) with the predominant species being 5-methylTHF, 29.3 ± 33.2% of which was monoglutamylated. Collectively, these data suggest that the quantity and form of folate (monoglutamylated) in the large intestine of human infants and piglets are sufficiently large to potentially affect folate status. J. Nutr. 134: 1389-1394, 2004.