To study the role of carbohydrates, in particular glucose, glucose 6-phosphate and mannose, as carbon substrates for extra- and intracellular replication of facultative intracellular enteric bacteria, mutants of two enteroinvasive Escherichia coli (EIEC) strains and a Salmonella enterica serovar Typhimurium isolate were constructed that were defective in the uptake of glucose and mannose (DeltaptsG, manXYZ), glucose 6-phosphate (DeltauhpT) or all three carbohydrates (DeltaptsG, manXYZ, uhpT). The ability of these mutants to grow in RPMI medium containing the respective carbohydrates and in Caco-2 cells was compared with that of the corresponding wild-type strains. In the three strains, deletions of ptsG, manXYZ or uhpT resulted in considerably different levels of inhibition of growth in vitro in the presence of glucose, mannose and glucose 6-phosphate, respectively, but hardly reduced their capability for intracellular replication in Caco-2 cells. Even the triple mutants DeltaptsG, manXYZ, uhpT of the three enterobacterial strains were still able to replicate in Caco-2 cells, albeit at strain-specific lower rates than the corresponding wild-type strains.