Young infants are highly susceptible to systemic dissemination of enteric pathogens such as Salmonella typhimurium when compared with older individuals. The mechanisms underlying this differential susceptibility have not been defined clearly. To better understand this phenomenon, we examined the responses of adult mice and preweaned pups to oral infection by S. typhimurium. We found clear age-specific differences, namely, an attenuated intestinal inflammatory response and a higher systemic bacterial burden in the pups compared with the adults. To elucidate the molecular basis for these differences, we obtained a microarray-based profile of gene expression in the small intestines of uninfected adult and preweaned animals. The results indicated a striking age-dependent increase in the intestinal expression of a number of IFN-γ-regulated genes involved in antimicrobial defense. This finding was confirmed by real-time quantitative PCR, which also demonstrated an age-dependent increase in intestinal expression of IFN-γ. The developmental up-regulation of the IFN-γ-regulated genes was dependent on both IFN-γ and a normal commensal microflora, as indicated by experiments in IFN-γ-knockout mice and germfree mice, respectively. However, the increase in expression of IFN-γ itself was independent of the commensal flora. The functional importance of IFN-γ in the immunological maturation of the intestine was confirmed by the observation that the response of adult IFN-γ-knockout animals to S. typhimurium infection resembled that of the wild-type pups. Our findings thus reveal a novel role for IFN-γ in the developmental regulation of antimicrobial responses in the intestine.