Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n ؍ 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n ؍ 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n ؍ 27] or 8 g/liter [PGL8 group; n ؍ 25]). A parallel breast-fed group (BF group) (n ؍ 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.Nondigestible food ingredients called prebiotics pass into the lower gastrointestinal tract and, by definition, may be selectively metabolized by mutualistic microorganisms, such as Lactobacillus spp. and Bifidobacterium spp., which in turn contribute to improved host health (12, 34). After lactose and lipids, oligosaccharides, which have prebiotic activity, are the third largest component of human breast milk (5 to 10 g/liter), and there are as many as 200 distinct molecular structures (5, 26). Lactobacilli and bifidobacteria are the predominant bacteria in the intestinal microbiota of breast-fed infants, whereas infants who receive cow's milk-based infant formulas, which naturally contain low levels of oligosaccharides, often have higher concentrations of potentially pathogenic bacteria, such as Enterobacteriaceae and clostridia, in their intestinal microbiota (4,15,17).Clinical investigations of infant formulas supplemented with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) at a range of concent...