Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored when searching for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium (S. Typhimurium), where they perform the third step in the purine synthesis. Surprisingly the results of the current study demonstrated that single gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection index < 0.03) in mouse infections. While the ΔpurT mutant multiplied as fast as the wild type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurN mutant was reduced by approximately 50 % in 20 hours. The attenuation of the ΔpurT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl-tetrahydrofolate by PurU. In the process of further characterization, we disclosed that in vivo the enzyme-complex GCV was the most important for formation of C-1 units in vivo (CI: 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C-1 units in vitro The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C-1 units and glycine by GlyA was not sufficient to ensure C-1 formation in S. Typhimurium in vivo. The study calls for re-investigations of the concept of metabolic redundancy in S. Typhimurium in vivo.